Use of G-CSF dimers in the treatment or prevention of chemotherapy or radiotherapy-induced neutropenia

Abstract

This application provides a method for treating or preventing conditions characterized by decreased leukocyte production in an individual (e.g., a human individual such as an individual with cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer simultaneously with or after the administration of a chemotherapeutic agent or radiotherapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to PCT / CN2023 / 105090, filed June 30, 2023, the entirety of which is incorporated herein by reference.

[0002] Reference to electronic sequence listings The entire contents of the electronic sequence listing (720622002041SEQLIST.xml, size: 34,709 bytes, created: June 20, 2024) are incorporated herein by reference.

[0003] The present invention relates to a method for treating or preventing conditions characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., a human individual such as an individual with cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer simultaneously with or after the administration of a chemotherapeutic agent or radiotherapy. [Background technology]

[0004] Cytotoxic chemotherapy remains one of the primary treatments for cancer. The biggest drawback of chemotherapy is that it indiscriminately kills healthy cells that rapidly proliferate and differentiate, even if they are tumor cells. The toxicity caused by chemotherapy is primarily reflected in the hematopoietic system and is clinically known as chemotherapy-induced neutropenia. Neutropenia is defined as a peripheral blood neutrophil count of 1.8 × 10⁶ in adults. 9 Less than / L, 1.5 × 10 in children 9 It is characterized by a neutropenia of less than / L. Neutropenia is often a precursor to infection, and the lower the neutrophil count, the higher the risk of infection. Neutropenia can also delay the next treatment cycle and directly affect the effectiveness of chemotherapy.

[0005] Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used as standard supportive therapy for chemotherapy-induced and / or radiotherapy-induced neutropenia in cancer patients treated with chemotherapy.

[0006] G-CSF is a hematopoietic glycoprotein produced by stromal cells, macrophages, endothelial cells, fibroblasts, and monocytes. G-CSF binds to the G-CSF receptor (G-CSFR) expressed on precursor cells in the bone marrow, promoting the proliferation and differentiation of various types of blood cells, such as neutrophils, which play a crucial role in the body's defense against bacterial infections. G-CSF can also participate in the immune response by activating mature neutrophils, or it can exert hematopoietic function by acting synergistically with other hematopoietic growth factors, such as stem cell factors, Flt-3 ligands, and GM-CSF.

[0007] G-CSF drugs such as filgrastim (Neupogen®) and its biosimilars, filgrastim-aafi (Nivestym®), filgrastim-sndz (Zarxio®), and filgrastim-ayow (Releuko®), are used to manage neutropenia in patients undergoing chemotherapy. However, these filgrastim or similar drugs have a short half-life of 3.5 hours and therefore need to be administered daily. Other G-CSF formulations, such as pegfilgrastim (e.g., Neulasta®) and efrapegrastim (e.g., ROLVEDON®), have half-lives extended to 30-50 hours through pegylation technology or fusion protein strategies, allowing them to be administered once per chemotherapy cycle. Currently, the recommended dosing regimen for both efrapegrastim (Rolontis®, HM10460A) and pegfilgrastim is administered the day after cytotoxic chemotherapy, which means that patients, who are usually weakened and uncomfortable after chemotherapy, will need to be transferred back to the hospital.

[0008] Burris et al. (J Oncol Pract. 2010;6(3):133-40) conducted a randomized clinical trial to compare data on the duration of severe (grade 4) neutropenia and the incidence of febrile neutropenia in patients receiving chemotherapy administered on the same day as pegfilgrastim versus chemotherapy administered 24 hours after chemotherapy. The results showed that the mean duration of severe neutropenia in cycle 1 was 1.2 days and 0.9 days longer in the same-day group compared to the next-day group in the breast cancer and lymphoma trials, respectively. Furthermore, in the breast and lymphoma trials, the absolute neutrophil count profile in same-day patients was earlier, deeper, and longer compared to patients receiving chemotherapy the next day. The conclusion of this trial confirmed that it is recommended to administer pegfilgrastim 24 hours after completion of chemotherapy in patients receiving pegfilgrastim with chemotherapy.

[0009] There is a need for more flexible drug regimens and / or superior G-CSF derivatives that reduce the burden on patients while providing equivalent or better efficacy in the treatment of neutropenia.

[0010] The contents of each publication, patent, patent application, and published patent application referenced herein are incorporated herein by reference in their entirety. [Overview of the Initiative]

[0011] One aspect of this application provides a method for treating or preventing a condition characterized by reduced leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring such treatment (e.g., an individual with cancer, e.g., a human individual), the method comprising administering an effective amount of granulocyte colony-stimulating factor (G-CSF) dimer to the individual (e.g., a human individual) less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later). In some embodiments, conditions characterized by decreased leukocyte production include one or more of the following: chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, decreased hematopoietic function, decreased immune function, decreased neutrophil count, decreased neutrophil recruitment, mobilization of peripheral blood progenitor cells, sepsis, infection, leukopenia, low bone marrow engraftment during transplantation, decreased bone marrow recovery in the treatment of myeloplasty or myelosuppression induced by radiation, chemicals or chemotherapy, radiotherapy-induced myeloplasty or myelosuppression, and acquired immunodeficiency syndrome. In some embodiments, conditions characterized by decreased leukocyte production are chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.

[0012] In some embodiments of any of the methods described above, the G-CSF dimer is administered within approximately 5 hours after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered within approximately 3 hours after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered within approximately 2 hours after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered within approximately 0.5 hours after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered simultaneously with the administration of the chemotherapeutic agent or radiotherapy.

[0013] In some embodiments of any of the methods described above, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. In some embodiments, an individual (e.g., a human individual) is administered two or more chemotherapeutic agents, including i) epirubicin and cyclophosphamide, ii) docetaxel and cyclophosphamide, iii) doxorubicin and cyclophosphamide, iv) docetaxel and doxorubicin, v) docetaxel, doxorubicin, and cyclophosphamide, or vi) cyclophosphamide, doxorubicin, and vincristine. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially. In some embodiments, when two or more chemotherapeutic agents are administered sequentially to an individual (e.g., a human individual), the G-CSF dimer is administered less than 24 hours after the administration of the last chemotherapeutic agent (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later).

[0014] In some embodiments of any of the methods described above, an individual (e.g., a human individual) is administered a chemotherapeutic agent or radiotherapy to treat cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma. In some embodiments, the cancer is breast cancer.

[0015] In some embodiments of any of the methods described above, an individual (e.g., a human individual) is administered at least four cycles of chemotherapy or radiotherapy, and the G-CSF dimer is administered less than 24 hours after the administration of the chemotherapy or radiotherapy in cycle 1. In some embodiments, the method further includes administering an effective amount of G-CSF dimer at approximately 24 hours after the administration of the chemotherapy or radiotherapy in at least each of cycles 2 to 4 (e.g., at 24 hours, 24 hours later, 48 hours later, or 48 hours later). In some embodiments, the chemotherapy or radiotherapy is administered on day 1 of each cycle. In some embodiments, each cycle is approximately 21 days long.

[0016] In some embodiments of any of the methods described above, an individual (e.g., a human individual) is administered at least four cycles of chemotherapy or radiotherapy, and the G-CSF dimer is administered less than 24 hours after the administration of the chemotherapy or radiotherapy in each of the four cycles. In some embodiments, the chemotherapy or radiotherapy is administered on day 1 of each cycle. In some embodiments, each cycle is approximately 21 days long.

[0017] In some embodiments of any of the methods described above, the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer and a dimerization domain. In some embodiments, the G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, within each monomeric subunit, the G-CSF monomer is connected to the dimerization domain via an optional linker. In some embodiments, the linker is approximately 6 to 30 amino acids long. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the dimerization domain within each monomeric subunit comprises at least two cysteine ​​molecules capable of forming an intermolecular disulfide bond. In some embodiments, the dimerization domain comprises at least a portion of an Fc fragment. In some embodiments, the Fc fragment comprises CH2 and CH3 domains. In some embodiments, the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or fragments or variants thereof. In some embodiments, the Fc fragment contains the amino acid sequence of any of SEQ ID NOs: 2-5. In some embodiments, the G-CSF monomer is located at the C-terminal end of the dimerization domain within each monomer subunit. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomer subunit. In some embodiments, each monomer subunit contains the amino acid sequence of any of SEQ ID NOs: 6-10, or a variant thereof having at least about 90% sequence identity with any of SEQ ID NOs: 6-10. In some embodiments, each monomer subunit contains the amino acid sequence of any of SEQ ID NOs: 6-10, for example, SEQ ID NO: 6. In some embodiments, the G-CSF dimer is efbemalenograstim alpha.

[0018] In some embodiments of any of the methods described above, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0019] In some embodiments of any of the methods described above, the individual is a human individual, for example, a human with cancer. In some embodiments, the individual has chemotherapy-induced neutropenia. In some embodiments, the individual has radiotherapy-induced neutropenia. In some embodiments, the G-CSF dimer is administered at a dose of about 5 mg to about 25 mg per dose, for example, about 10 mg to about 25 mg per dose, or about 20 mg per dose.

[0020] In some embodiments of any of the methods described above, the G-CSF dimer is administered subcutaneously. [Brief explanation of the drawing]

[0021] [Figure 1] This is a diagram illustrating the structure of an exemplary G-CSF dimer (e.g., efbemalenograstim alfa). In the diagram, "-" represents a linker, the oval object labeled "G-CSF" represents a G-CSF monomer, and the oval object labeled "Fc" represents an Fc fragment (e.g., human IgG-derived Fc). The exemplary G-CSF dimer contains two monomeric subunits. Each monomeric subunit contains a G-CSF monomer, a linker, and an Fc fragment from N' to C'. [Figure 2]This study demonstrates the effects of efbemarenograstim alfa and pegfilgrastim on absolute neutrophil count (ANC) after acute TC (docetaxel + cyclophosphamide)-induced neutropenia in SD rats. Efbemarenograstim alfa or pegfilgrastim was administered 0 hours (i.e., concurrently with TC treatment), 2 hours, 5 hours, or 24 hours after TC chemotherapy. Rats not receiving TC chemotherapy were used as "normal" controls. Rats receiving TC chemotherapy and vehicle treatment were also used as controls. [Modes for carrying out the invention]

[0022] Current dosing regimens for approved G-CSF formulations prevent patients from receiving G-CSF treatment to counteract chemotherapy-induced neutropenia until 24 hours or even 48 hours after cytotoxic chemotherapy. Such dosing regimens necessitate that patients, weakened and unwell after chemotherapy, be transported back to the hospital within the next 1-2 days. The present invention provides a "same-day dosing" regimen for long-acting G-CSF dimers, which allows patients to receive G-CSF treatment less than 24 hours after chemotherapy or radiotherapy, for example, less than 6 hours, for example, 0 hours (i.e., concurrently), 0.5 hours, 3 hours, or 5 hours after chemotherapy or radiotherapy. The methods described herein not only reduce the burden on patients but also provide equivalent or even superior efficacy in the treatment of neutropenia compared to long-acting G-CSF formulations, such as pegfilgrastim (Neulasta®).

[0023] Accordingly, in some embodiments, methods are provided for treating or preventing conditions characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in individuals requiring treatment (e.g., humans, e.g., individuals with cancer), the methods comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (i.e., simultaneously with administration), or 0.5 hours, 2 hours, 3 hours, or 5 hours later).

[0024] I. Definition As used herein, the term “absolute neutrophil count (ANC)” is a measure of the number of neutrophil granulocytes present in the blood. Neutrophils are a type of white blood cell that plays a central role in the immune system’s defense against infections, particularly bacterial infections. They are the most abundant type of white blood cell in most mammals and form an integral part of the innate immune system.

[0025] As used herein, “myelosuppression” refers to the suppression of one or more components of hematopoiesis, which manifests as an abnormal level of one or more of the cell types that are products of this process. For an overview of hematopoiesis and the properties of hematopoietic cells, see Clinical Immunology: Principles and Practice, Vol. 1, Ch. 2, pp. 15-24 (Lewis and Harriman, eds. Mosby-Year Book, Inc. 1996), which are incorporated herein by reference. At a general level, myelosuppression refers to a decrease in the number of white blood cells (WBCs) and / or platelets. At a more specific level, it also refers to the suppression of one or more of the following cells resulting from hematopoiesis: B cells, T cells, natural killer cells, dendritic cells, macrophages, neutrophils, eosinophils, basophils, mast cells, and platelets. In contrast, “myelosuppression” is therefore the opposite of myelosuppression. “Myelosuppressant” or “myelosuppressive therapy” refers to a drug or treatment that can induce myelosuppression.

[0026] As used herein, the term "neutropenia" is defined as a condition characterized by a decrease in the number of neutrophils. Normal is defined as a condition where the absolute neutrophil count is 2.0×10 9 / L or higher. Grade 1 is defined as neutropenia where the absolute neutrophil count is 1.5×10 9 / L to 1.999×10 9 / L. Grade 2 is defined as neutropenia where the absolute neutrophil count is 1.0×10 9 / L to 1.499×10 9 / L. Grade 3 is defined as neutropenia where the absolute neutrophil count is 0.5×10 9 / L to 0.999×10 9 / L. Grade 4 (severe neutropenia) is defined as neutropenia having an absolute neutrophil count less than 0.5×10 9 / L. The terms "severe neutropenia" and "Grade 4 neutropenia" may be used interchangeably.

[0027] The term "febrile neutropenia" (FN) is defined as a condition with neutropenia and a single oral temperature of 38.3°C (101°F) or higher, or a body temperature of 38.0°C (100.4°F) or higher persisting for 1 hour, and is usually classified as an ANC of less than 500 cells / mm3, or an ANC expected to decrease to less than 500 cells / mm3 within the next 48 hours. This is a serious complication commonly seen in people undergoing cancer treatment, especially chemotherapy.

[0028] The "targets" for administration are not limited to these, but include humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or non-human animals, such as primates (e.g., crab-eating macaques, rhesus macaques), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the individual is human. In certain embodiments, the individual is a non-human animal. The individual may be of any age and / or sex. In some embodiments, the individual has cancer.

[0029] Unless otherwise specified herein, the numbering of amino acid residues within the IgG Fc region follows the EU numbering system for antibodies, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0030] The "CH1 domain" (also referred to as the "C1" domain of the "H1" domain) typically extends from approximately amino acids 118 to 215 (EU numbering system). The "hinge region" is generally defined as the IgG region corresponding to Glu216-Pro230 in human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond in the same position. The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc domain typically extends from approximately amino acids 231 to 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. It is hypothesized that carbohydrates can provide an alternative for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985). The "CH3 domain" (also referred to as the "C3" domain) includes a stretch from the C-terminal residue of the Fc domain to the CH2 domain (i.e., from approximately amino acid residue 341 to the C-terminus of the antibody sequence, typically to amino acid residue 446 or 447 of IgG).

[0031] In this specification, the terms “Fc domain” or “fragmentary crystalline region” are used to define the C-terminal region of an immunoglobulin heavy chain, and include both the native Fc domain and variant Fc domains. While the boundaries of the Fc domain of an immunoglobulin heavy chain can vary, the Fc domain of a human IgG heavy chain is typically defined as extending from the amino acid residue position Cys226 or Pro230 to its carboxyl terminus. The lysine at the C-terminus of the Fc domain (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an Fc-containing protein, or by genetic engineering of the nucleic acid encoding the Fc-containing protein. Thus, compositions of Fc-containing proteins may include populations of Fc-containing proteins in which all K447 residues have been removed, populations of Fc-containing proteins in which the K447 residue has not been removed, and populations of Fc-containing proteins having mixtures of Fc-containing proteins with and without the K447 residue. Natural sequence Fc domains suitable for use in Fc-containing proteins described herein may include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0032] In this specification, “amino acid sequence identity percentage (%)” or “homology” for polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the polypeptide being compared, after the sequences have been aligned considering all conserved substitutions as part of sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage (%) can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for evaluating alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the value of amino acid sequence identity percentage is generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0033] As used herein, the terms “pharmaceutical preparation” or “pharmaceutical composition” mean a preparation in which the biological activity of the active ingredients contained herein is effective and which does not contain any ingredients that are to an extent unacceptable to the subject to which the preparation is administered.

[0034] A “sterile” preparation is either sterile, free from or essentially free from viable microorganisms and their spores.

[0035] The terms “disease,” “disorder,” and “condition” are used interchangeably in this specification.

[0036] As used herein, “treatment” or “treating” refers to an approach to obtain beneficial or desirable outcomes, including clinical outcomes. For the purposes of this application, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms of a disease (e.g., fever, infection, decreased ANC); reducing the severity of the disease (e.g., lowering the grade of neutropenia); reducing the duration of the disease; stabilizing the disease (e.g., preventing or delaying disease exacerbation); preventing or delaying disease relapse; delaying or slowing disease progression (e.g., progression to the next grade of neutropenia or febrile neutropenia); improving the disease state; achieving (partial or complete) remission of the disease; reducing the dose of one or more other medicines required to treat the disease; delaying disease progression; improving or enhancing quality of life; increasing weight gain; and / or extending survival. Furthermore, “treatment” also encompasses the reduction of pathological outcomes (e.g., fever, infection, decreased ANC), including the severity and incidence of pathological outcomes. The methods of this application conceivable one or more of these forms of treatment. If the methods are for treating cancer, the methods in some embodiments may also prevent or delay the spread of cancer (e.g., metastasis) and / or reduce pathological outcomes (e.g., tumor volume).

[0037] As used herein, the terms “effective dose,” “pharmaceutical effective dose,” and “therapeutic effective dose” refer to an amount of a drug that is non-toxic but sufficient to produce a desired biological outcome. This outcome may be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or mitigation of signs, symptoms, or causes of a disease or disorder, or any other desirable change in a biological system. As will be recognized by those skilled in the art, the effective dose of a drug may vary depending on the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and factors such as the age, weight, health status, and condition of the subject.

[0038] As used herein, “reference” means any sample, standard, or level used for comparative purposes. References may be obtained from healthy and / or non-disease samples. In some cases, references may be obtained from untreated samples. In some cases, references may be obtained from non-disease or untreated samples of individuals. In some cases, references may be obtained from one or more healthy individuals that are not individuals or patients.

[0039] As used herein, “delay the onset of disease” means to postpone, prevent, slow, delay, stabilize, suppress, and / or postpone the onset of disease. This delay may be of varying lengths depending on the medical history and / or the individual being treated. As will be obvious to those skilled in the art, a sufficient or substantial delay may substantially encompass prevention in that the individual does not develop the disease.

[0040] As used herein, “prevention” includes providing prevention with respect to the onset or recurrence of a disease in an individual who may be predisposed to the disease but has not yet been diagnosed with the disease.

[0041] It should be understood that embodiments of the present invention described herein include embodiments that "consist of" and / or "essentially consist of."

[0042] In this specification, any reference to a value or parameter that is "about" includes (and shall be described) variations of that value or parameter itself. For example, a statement that refers to "about X" includes a statement of "X".

[0043] Where used herein, a reference to "not" a certain value or parameter generally means and describes "other than" a certain value or parameter. For example, "a method is not used to treat type X cancer" means "a method is used to treat type X cancer."

[0044] As used herein, the term "approximately X to Y" has the same meaning as "from approximately X to approximately Y".

[0045] As used herein and in the appended claims, the singular forms "a," "or," and "the" refer to multiple subjects unless otherwise explicitly indicated in the content.

[0046] II. Methods for treating or preventing conditions caused by G-CSF dimers In some embodiments, methods are provided for treating or preventing conditions characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in individuals requiring treatment (e.g., humans, e.g., individuals with cancer), the methods comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later). In some embodiments, the conditions characterized by decreased leukocyte production are chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, methods are provided for increasing one or more of the following in an individual requiring assistance (e.g., a human, e.g., an individual with cancer): i) absolute neutrophil count (ANC), ii) granulocyte count (e.g., in a subject eligible for bone marrow transplantation), iii) stem cell production, iv) hematopoiesis, and v) hematopoietic progenitor cell (HPC) count (e.g., by at least about any of 10%, 20%, 50%, 70%, 90%, 1x, 2x, 5x, 20x, or more), the method comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy to the individual. In some embodiments, methods are provided for reducing the duration of chemotherapy-induced neutropenia (e.g., grade 4) or radiotherapy-induced neutropenia (e.g., grade 4) in an individual in need (e.g., a human, e.g., an individual with cancer) (e.g., reducing it by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more), the method comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy to the individual.In some embodiments, methods are provided to reduce (e.g., reduce by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevent the incidence of chemotherapy-induced neutropenia (e.g., grade 4) or radiotherapy-induced neutropenia (e.g., grade 4) in individuals in need (e.g., humans, e.g., individuals with cancer), the method comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy to the individual. In some embodiments, the method involves reducing the incidence of about 0.5 × 10⁻¹⁰ in the individual. 9 The duration of ANC below 0.5 × 10⁻¹⁰ hours is reduced to less than approximately 24 hours (e.g., less than approximately 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.2, 0.1 hours, or less). In some embodiments, this method reduces the individual's ANC to approximately 0.5 × 10⁻¹⁰ hours. 9 Less than / L (for example, at least about 0.4 × 10) 9 / L, 0.2×10 9 / L, 0.1×10 9 / L, 1.0×10 7 To prevent the ANC from reaching less than 10 / L or less. In some embodiments, administration of G-CSF dimer reduces the ANC to approximately 0.5 × 10⁻⁶. 9 Within approximately 10 days from the first occurrence of less than / L (for example, within approximately 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less), approximately 2.0 × 10 9Increase to over / L. In some embodiments, methods are provided to reduce or prevent the incidence of febrile neutropenia (FN) in individuals in need (e.g., humans, e.g., individuals with cancer) (e.g., reduce by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more), the method comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy to the individual. In some embodiments, methods are provided for activating one or more of the following in an individual requiring assistance (e.g., a human, e.g., an individual with cancer): i) neutrophil progenitor cells, ii) bone marrow stem cells, and iii) mature neutrophils, the method comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy to the individual. In some embodiments, the G-CSF dimer is administered within approximately 5 hours after administration of the chemotherapeutic agent or radiotherapy (e.g., at 5 hours). In some embodiments, the G-CSF dimer is administered within approximately 3 hours after administration of the chemotherapeutic agent or radiotherapy (e.g., at 3 hours). In some embodiments, the G-CSF dimer is administered within approximately 2 hours after administration of the chemotherapeutic agent or radiotherapy (e.g., at 2 hours). In some embodiments, the G-CSF dimer is administered within approximately 0.5 hours after administration of the chemotherapeutic agent or radiotherapy (e.g., at 0.5 hours). In some embodiments, the G-CSF dimer is administered concurrently with the administration of a chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit.In some embodiments, the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered in doses of about 5 mg to about 25 mg, for example, about 20 mg per dose. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered a chemotherapeutic agent or radiotherapy for the treatment of cancer (e.g., breast cancer). In some embodiments, the individual is administered two chemotherapeutic agents: docetaxel and cyclophosphamide, for example, simultaneously (e.g., in the same formulation or in separate formulations).

[0047] Accordingly, in some embodiments, methods are provided for treating or preventing conditions characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual in need (e.g., a human, e.g., an individual with cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer simultaneously with or after the administration of a chemotherapeutic agent or radiotherapy, the method comprising administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), the G-CSF dimer comprising two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, methods are provided for treating or preventing conditions characterized by leukocyte deficiency (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in individuals requiring treatment (e.g., humans, e.g., humans with cancer), the method comprising administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), the G-CSF dimer comprising two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, methods are provided for treating or preventing conditions characterized by leukocyte deficiency (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in individuals requiring treatment (e.g., humans, e.g., humans with cancer), wherein the method involves administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later). The G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. The G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20. The pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at a dose of about 5 mg to about 25 mg, for example, about 20 mg per dose. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered a chemotherapeutic agent or radiotherapy for the treatment of cancer (e.g., breast cancer). In some embodiments, the individual is administered two chemotherapeutic agents: docetaxel and cyclophosphamide, for example, simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, the G-CSF dimer is efbemarenograstim alfa.

[0048] In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising administering an effective dose of a chemotherapeutic agent or radiotherapy to the individual, and further administering an effective dose of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., fbemarenograstim alfa) to the individual less than 24 hours after the administration of the effective dose of the chemotherapeutic agent or radiotherapy, wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection). In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents: docetaxel and cyclophosphamide are administered to the individual, for example, simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, administration of G-CSF dimers increases one or more of the following: i) absolute neutrophil count (ANC), ii) granulocyte count (e.g., in subjects eligible for bone marrow transplantation), iii) stem cell production, iv) hematopoiesis, and v) the number of hematopoietic progenitor cells (HPCs) in the individual (e.g., an increase of at least about 10%, 20%, 50%, 70%, 90%, 1x, 2x, 5x, 20x, or more). In some embodiments, administration of G-CSF dimers reduces the duration of chemotherapy-induced neutropenia (e.g., grade 4) or radiotherapy-induced neutropenia (e.g., grade 4) in the individual (e.g., a reduction of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, administration of G-CSF dimers reduces (e.g., reduces by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the incidence of chemotherapy-induced neutropenia (e.g., grade 4) or radiotherapy-induced neutropenia (e.g., grade 4) in individuals. In some embodiments, administration of G-CSF dimers reduces the incidence of about 0.5 × 10⁻⁶ in individuals. 9The duration of ANC below 0.5 × 10⁻¹⁶ / L is reduced to less than approximately 24 hours (e.g., less than approximately 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.2, 0.1 hours, or less). In some embodiments, administration of G-CSF dimer reduces the individual's ANC to approximately 0.5 × 10⁻¹⁶. 9 Less than / L (for example, at least about 0.4 × 10) 9 / L, 0.2×10 9 / L, 0.1×10 9 / L, 1.0×10 7 To prevent the ANC from reaching less than 10 / L or less. In some embodiments, administration of G-CSF dimer reduces the ANC to approximately 0.5 × 10⁻⁶. 9 Within approximately 10 days from the first occurrence of less than / L (for example, within approximately 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less), approximately 2.0 × 10 9Increase to over / L. In some embodiments, administration of G-CSF dimers reduces or prevents the incidence of FN in an individual (e.g., at least one of the following reductions: at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, administration of G-CSF dimers activates one or more of the following in an individual: i) neutrophil progenitor cells, ii) bone marrow stem cells, and iii) mature neutrophils.In some embodiments, a method for treating cancer involves the following biological activities, namely: (1) killing cancer cells (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), (2) inhibiting the proliferation of cancer cells (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), and (3) reducing tumor size (e.g., less (4) Reduction of one or more symptoms in an individual with cancer (e.g., by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), (5) Reduction of tumor metastases (e.g., metastases to lymph nodes) (e.g., by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), (6) Reduction of tumor metastases (e.g., metastases to lymph nodes) (e.g., by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%) (6) suppression of any of 10%, 80%, 90%, or 100%; (7) reduction of the incidence, occurrence, or burden of existing tumor metastases (e.g., metastases to lymph nodes) (e.g., by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%); (8) extension of survival, e.g., survival of an individual by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 48 months; (8) extension of time to cancer progression, e.g., extension of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 60 weeks, or more, and (9) prevention, inhibition, or reduction of the likelihood of cancer recurrence (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.In some embodiments, an individual is administered two or more chemotherapeutic agents, including i) epirubicin and cyclophosphamide, ii) docetaxel and cyclophosphamide, iii) doxorubicin and cyclophosphamide, iv) docetaxel and doxorubicin, v) docetaxel, doxorubicin, and cyclophosphamide, or vi) cyclophosphamide, doxorubicin, and vincristine. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially. In some embodiments, the two or more chemotherapeutic agents are administered in R-CHOP-like chemotherapy (R: rituximab; C: cyclophosphamide (e.g., CYTOXAN®); H: doxorubicin (e.g., ADRIAMYCIN®); O: vincristine (e.g., ONCOVIN®); P: prednisone). In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma. In some embodiments, the G-CSF dimer is administered within approximately 5 hours (e.g., 5 hours) after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered within approximately 3 hours (e.g., 3 hours) after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered within approximately 2 hours (e.g., 2 hours) after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered within approximately 0.5 hours (e.g., 0.5 hours) after administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer is administered concurrently with the administration of chemotherapeutic agents or radiotherapy. In some embodiments, the G-CSF dimer is administered with rituximab (e.g., 375 mg / m²). 2 ), two or more chemotherapy agents (e.g., 750 mg / m²) 2 Cyclophosphamide, 50 mg / m² 2 Doxorubicin, and 1.4 mg / m²2 Vincristine (up to 2.0 mg); and administered with prednisone or prednisolone (e.g., 100 mg). In some embodiments, rituximab (e.g., 375 mg / m²) is administered. 2 ) is administered on day 1; G-CSF dimer is administered on day 2 with two or more chemotherapeutic agents (e.g., 750 mg / m²). 2 Cyclophosphamide, 50 mg / m² 2 Xorubicin, and 1.4 mg / m² 2 Vincristine (up to 2.0 mg) is administered concurrently; prednisone or prednisolone (e.g., 100 mg) is administered daily from day 2 to day 6. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (e.g., SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located on the N-terminal side of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered in doses of about 5 mg to about 25 mg, for example, about 20 mg per dose.

[0049] In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising administering an effective dose of a chemotherapeutic agent or radiotherapy to the individual, and further administering an effective dose of a G-CSF dimer to the individual less than 24 hours after the administration of the effective dose of the chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection), the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising administering an effective dose of a chemotherapeutic agent or radiotherapy to the individual, and further administering an effective dose of a G-CSF dimer to the individual less than 24 hours after the administration of the effective dose of the chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection), and the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising administering an effective dose of a chemotherapeutic agent or radiotherapy to the individual, and further administering an effective dose of a G-CSF dimer to the individual less than 24 hours after the administration of the effective dose of the chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer is for treating a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia). To alleviate or prevent (disease, radiotherapy-induced neutropenia, or infection), the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. The G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20. The pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at approximately 5 mg to approximately 25 mg per dose, for example, approximately 20 mg per dose. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents: docetaxel and cyclophosphamide are administered to the individual, for example, simultaneously (for example, in the same formulation or in separate formulations). In some embodiments, the G-CSF dimer is efbemarenograstim alfa.

[0050] G-CSF dimers (e.g., G-CSF-Fc dimers, e.g., fbemalenograstim alfa) or their pharmaceutical compositions can be administered less than 24 hours after administration of a chemotherapeutic agent or radiotherapy to an individual, for example, simultaneously with the administration of the chemotherapeutic agent or radiotherapy (i.e., at 0 hours), or within any of the following times after the administration of the chemotherapeutic agent or radiotherapy: 0.1 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 23.5 hours. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered within approximately 0.1 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours after the administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered within approximately 0.1 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after the administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered within approximately 5 hours (e.g., 5 hours) after the administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered within approximately 3 hours (e.g., 3 hours) after the administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered within approximately 2 hours (e.g., 2 hours) after the administration of the chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered within approximately 0.5 hours (e.g., 0.5 hours) after the administration of a chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered simultaneously with (or at 0 hours after) the administration of a chemotherapeutic agent or radiotherapy. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered on the same day as the administration of a chemotherapeutic agent or radiotherapy.

[0051] In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of chemotherapeutic agents or radiotherapy, the administration of the G-CSF dimer or its pharmaceutical composition occurs less than 24 hours after the last dose of the chemotherapeutic agent or radiotherapy in the cycle (e.g., at 0 hours (i.e., simultaneously with administration), or 0.5 hours, 3 hours, or 5 hours later). For example, if the chemotherapeutic agent or radiotherapy is administered in one dose on day 1 of the cycle, the administration of the G-CSF dimer or its pharmaceutical composition occurs less than 24 hours after the first dose of the chemotherapeutic agent or radiotherapy in the cycle. If the chemotherapeutic agent or radiotherapy is administered in a first dose on day 1 of the cycle (i.e., a cycle of at least two days) and a second dose on day 2, the administration of the G-CSF dimer or its pharmaceutical composition occurs less than 24 hours after the second dose of the chemotherapeutic agent or radiotherapy in the cycle. Similarly, when an individual is administered two or more (e.g., four) doses of a chemotherapeutic agent or radiotherapy in only one cycle, the administration of the G-CSF dimer or its pharmaceutical composition is less than 24 hours after the last dose of the chemotherapeutic agent. In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of a chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or its pharmaceutical composition less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy is simultaneous in each cycle after the administration of the chemotherapeutic agent or radiotherapy, for example, all at 0 hours (simultaneous with administration), all at 0.5 hours, all at 3 hours, and all at 5 hours. In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of a chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or its pharmaceutical composition less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy may differ in two or more cycles, for example, 5 hours after the administration of the chemotherapeutic agent or radiotherapy in one cycle, and 0 hours (simultaneous with administration) in another cycle.In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of chemotherapeutic agents or radiotherapy, the administration of the G-CSF dimer or its pharmaceutical composition is less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in at least one cycle (e.g., at 0 hours (i.e., simultaneously with administration), or 0.5 hours, 3 hours, or 5 hours later). For the second and subsequent cycles of chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or its pharmaceutical composition may be 24 hours after the administration of the chemotherapeutic agent or radiotherapy (e.g., 24 hours, 24 hours later, 48 hours, or 48 hours later).

[0052] In some embodiments, an individual is administered two or more chemotherapeutic agents (e.g., docetaxel and cyclophosphamide), for example, two or more chemotherapeutic agents per cycle. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially, for example, the first chemotherapeutic agent is administered on day 1 and the second chemotherapeutic agent on day 2. When an individual is administered two or more chemotherapeutic agents sequentially, in some embodiments, the G-CSF dimer is administered less than 24 hours after the administration of the last chemotherapeutic agent, for example, the last chemotherapeutic agent of the cycle (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later). In some embodiments, if two or more chemotherapeutic agents are administered at least two days apart, for example, if the first chemotherapeutic agent is administered on day 1 and the second chemotherapeutic agent is administered on day 3 or later, depending on the ANC level and / or other condition of the individual being treated (e.g., infection, fever, vital signs, clinical laboratory values), the G-CSF dimer may be administered less than 24 hours after the administration of the first chemotherapeutic agent (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), or less than 24 hours after the administration of each of the two or more chemotherapeutic agents.

[0053] In some embodiments, the individual is administered at least two (e.g., two, three, or four) cycles of chemotherapy or radiotherapy. In some embodiments, the G-CSF dimer is administered less than 24 hours after the administration of the chemotherapy or radiotherapy in cycle 1. In some embodiments, the individual is administered at least four cycles of chemotherapy or radiotherapy, and the G-CSF dimer is administered less than 24 hours after the administration of the chemotherapy or radiotherapy in cycle 1. Accordingly, in some embodiments, a method is provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual in need (e.g., a human, e.g., an individual with cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer concurrently with or after the administration of a chemotherapeutic agent or radiotherapy, wherein the individual has received at least two (e.g., two, three, or four) cycles of chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle), and the method comprises administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (concurrent with administration), or 0.5 hours, 3 hours, or 5 hours later).In some embodiments, a method is provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual in need (e.g., a human, e.g., an individual with cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer concurrently with or after the administration of a chemotherapeutic agent or radiotherapy, wherein the individual is administered at least four cycles (e.g., four cycles) of the chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle), and the method comprises administering an effective amount of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (concurrent with administration), or 0.5 hours, 3 hours, or 5 hours later). In some embodiments, the individual is administered the chemotherapeutic agent or radiotherapy to treat cancer (e.g., breast cancer). In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least two (e.g., 2, 3, or 4) cycles; and ii) administering an effective amount of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemarenograstim alfa) to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocytosis (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the two or more cycles (e.g., cycle 1, or cycles 1-2).In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective amount of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocytosis (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the four or more cycles (e.g., cycle 1, or cycles 1-4). In some embodiments, the method further includes administering an effective amount of G-CSF dimer 24 hours after the administration of a chemotherapeutic agent or radiotherapy in each cycle starting from cycle 2, for example, in each cycle from cycles 2 to 4 (e.g., 24 hours, or 26, 28, 30, 32, 36, 40, 44, 48, 60 hours, or any of these). In some embodiments, the G-CSF dimer is administered approximately 48 hours after the administration of a chemotherapeutic agent or radiotherapy in each cycle starting from cycle 2, for example, in each cycle from cycles 2 to 4 (e.g., 48 hours, or 48 hours). In some embodiments, the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is approximately 21 days long. In some embodiments, the timing of G-CSF dimer administration is the same in each cycle starting from cycle 2, for example, in each cycle from cycles 2 to 4. In some embodiments, the administration time of the G-CSF dimer differs in two or more cycles starting from cycle 2, for example, in at least two or more cycles from cycles 2 to 4.In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents, docetaxel and cyclophosphamide, are administered to the individual in each cycle, for example, simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered in doses of about 5 mg to about 25 mg, for example, about 20 mg per dose.In some embodiments, administration of a G-CSF dimer or its pharmaceutical composition in one or more cycles (e.g., cycle 1, cycles 1-2, cycles 2-4, or cycles 1-4) has the following effects, namely: i) a reduction in the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; ii) a reduction or prevention of the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; iii) a reduction or prevention of the incidence of FN; iv) an increase in ANC; v) activation of neutrophil progenitor cells; vi) activation of bone marrow stem cells; vii) activation of mature neutrophils; viiii) an increase of approximately 0.5 × 10⁻¹⁶ in an individual. 9 A reduction in the duration of ANC below / L to approximately less than 24 hours, ix) ANC of an individual is approximately 0.5 × 10 9 To prevent it from reaching less than / L, x) ANC is approximately 0.5 × 10 9 From the first occurrence of less than / L, approximately 2.0 × 10 within about 10 days 9 Achieve one or more of the following increases: / L or higher. In some embodiments, chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is grade 4 neutropenia.

[0054] In some embodiments, methods are provided for treating or preventing a condition characterized by reduced leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring treatment (e.g., a human, e.g., an individual with cancer), wherein the individual is administered at least four cycles (e.g., four cycles) of chemotherapeutic agents or radiotherapy (e.g., the chemotherapeutic agents or radiotherapy are administered on day 1 of each cycle), and the method comprises administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), 0.5 hours, 3 hours, or 5 hours later), wherein the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2.In some embodiments, methods are provided for treating or preventing a condition characterized by reduced leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring treatment (e.g., a human, e.g., an individual with cancer), wherein the individual is administered at least four cycles (e.g., four cycles) of chemotherapeutic agents or radiotherapy (e.g., the chemotherapeutic agents or radiotherapy are administered on day 1 of each cycle), and the method comprises administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, methods are provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring treatment (e.g., a human, e.g., an individual with cancer), wherein the individual is administered at least four cycles (e.g., four cycles) of chemotherapy or radiotherapy (e.g., the chemotherapy or radiotherapy is administered on day 1 of each cycle), and the method is performed less than 24 hours (e.g., 0 hours) after administration of the chemotherapy or radiotherapy in cycle 1. The method involves administering an effective amount of G-CSF dimer to an individual (simultaneously with administration, or 0.5 hours, 3 hours, or 5 hours later), wherein the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6, and the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously.In some embodiments, the G-CSF dimer is administered at a dose of approximately 5 mg to approximately 25 mg per dose, for example, approximately 20 mg per dose. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered a chemotherapeutic agent or radiotherapy for the treatment of cancer (e.g., breast cancer). In some embodiments, the individual is administered two chemotherapeutic agents, docetaxel and cyclophosphamide, for example, simultaneously in each cycle (e.g., in the same formulation or in separate formulations). In some embodiments, the method further includes administering an effective dose of the G-CSF dimer 24 hours after the administration of the chemotherapeutic agent or radiotherapy in at least each cycle of cycles 2 to 4 (e.g., 24 hours later, or after 26, 28, 30, 32, 36, 40, 44, 48, 60 hours, or any later). In some embodiments, the G-CSF dimer is administered approximately 48 hours after the administration of the chemotherapeutic agent or radiotherapy in at least each cycle 2–4 (e.g., 48 hours or 48 hours after). In some embodiments, the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle (e.g., at least two days per cycle). In some embodiments, each cycle is approximately 21 days long. In some embodiments, the administration time of the G-CSF dimer is the same in at least each cycle 2–4. In some embodiments, the administration time of the G-CSF dimer differs in at least two or more cycles of cycles 2–4. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.

[0055] In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), and G-CSF Administration of the dimer reduces or prevents a condition characterized by decreased leukocyte production in at least one of four or more cycles (e.g., cycle 1, or cycles 1-4) (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection), wherein the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, an individual is administered a chemotherapeutic agent or radiotherapy for the treatment of cancer (e.g., breast cancer).In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective amount of a G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer mitigates or prevents a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the four or more cycles (e.g., cycle 1, or cycles 1-4), the G-CSF dimer comprising two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective amount of a G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in cycle 1 (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer is performed in at least one of the four or more cycles (e.g., The G-CSF dimer is formulated into a pharmaceutical composition comprising (a) approximately 20 mg / mL of G-CSF dimer, (b) approximately 10 mM sodium acetate, (c) approximately 1 mM EDTA, (d) approximately 5% (w / v) sorbitol, and (e) approximately 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of approximately 5.2.In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at a dose of about 5 mg to about 25 mg per dose, for example, about 20 mg per dose. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents, docetaxel and cyclophosphamide, are administered to the individual in each cycle, for example, simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, the method further includes administering an effective amount of G-CSF dimer 24 hours after the administration of the chemotherapeutic agent or radiotherapy in at least each cycle of cycles 2 to 4 (e.g., 24 hours, or 26, 28, 30, 32, 36, 40, 44, 48, 60 hours, or any later). In some embodiments, the G-CSF dimer is administered approximately 48 hours after the administration of the chemotherapeutic agent or radiotherapy in at least each cycle of cycles 2 to 4 (e.g., 48 hours, or 48 hours later). In some embodiments, the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is approximately 21 days long. In some embodiments, the administration time of the G-CSF dimer is the same in at least each cycle of cycles 2 to 4. In some embodiments, the administration time of the G-CSF dimer differs in at least two or more cycles of cycles 2 to 4. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.

[0056] In some embodiments, the individual is administered at least two cycles (e.g., two, three, or four cycles) of chemotherapeutic agents, and the G-CSF dimer is administered less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in each of the at least two cycles. In some embodiments, the individual is administered at least four cycles of chemotherapeutic agents or radiotherapy, and the G-CSF dimer is administered less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in each of the at least four cycles. Accordingly, in some embodiments, a method is provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual in need (e.g., a human, e.g., an individual with cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer concurrently with or after the administration of a chemotherapeutic agent or radiotherapy, wherein the individual is administered at least two (e.g., two, three, or four) cycles of chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day one of each cycle), and the method comprises administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in each of the at least two cycles (e.g., 0 hours (concurrent with administration), or 0.5 hours, 3 hours, or 5 hours later).In some embodiments, a method is provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual in need (e.g., a human, e.g., an individual with cancer), by administering a granulocyte colony-stimulating factor (G-CSF) dimer concurrently with or after the administration of a chemotherapeutic agent or radiotherapy, wherein the individual receives at least four cycles (e.g., four cycles) of chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle), and the method comprises administering an effective amount of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy in each of the at least four cycles (e.g., 0 hours (concurrent with administration), or 0.5 hours, 3 hours, or 5 hours later). In some embodiments, an individual is administered a chemotherapy agent or radiotherapy to treat cancer (e.g., breast cancer). In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., a human, e.g., an individual with cancer), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least two (e.g., two, three, or four) cycles; and ii) administering an effective amount of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in each of the at least two cycles (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocytosis (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the two or more cycles (e.g., cycle 1, or cycles 1-2).In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., a human, e.g., an individual with cancer), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective amount of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in each of the at least four cycles (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer reduces or prevents a condition characterized by decreased leukocytosis (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the four or more cycles (e.g., cycle 1, or cycles 1-4). In some embodiments, the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is approximately 21 days long. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of at least two cycles. In some embodiments, the administration time of the G-CSF dimer differs in each cycle of at least two cycles. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of at least four cycles. In some embodiments, the administration time of the G-CSF dimer differs in two or more cycles (e.g., four) of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 5 hours (e.g., 5 hours) after the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least two cycles, e.g., each cycle of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 3 hours (e.g., 3 hours) after the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least two cycles, e.g., each cycle of at least four cycles.In some embodiments, the G-CSF dimer is administered within approximately 0.5 hours (e.g., at 0.5 hours) after the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least two cycles, for example, in each cycle of at least four cycles. In some embodiments, the G-CSF dimer is administered concurrently with (or at 0 hours) the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least two cycles, for example, in each cycle of at least four cycles. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents, docetaxel and cyclophosphamide, are administered to the individual in each cycle, for example, concurrently (e.g., in the same formulation or in separate formulations). In some embodiments, the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered in doses of about 5 mg to about 25 mg, for example, about 20 mg per dose.In some embodiments, administration of a G-CSF dimer or its pharmaceutical composition in one or more cycles (e.g., cycle 1, cycles 1-2, cycles 2-4, or cycles 1-4) has the following effects, namely: i) a reduction in the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; ii) a reduction or prevention of the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; iii) a reduction or prevention of the incidence of FN; iv) an increase in ANC; v) activation of neutrophil progenitor cells; vi) activation of bone marrow stem cells; vii) activation of mature neutrophils; viiii) an increase of approximately 0.5 × 10⁻¹⁶ in an individual. 9 A reduction in the duration of ANC below / L to approximately less than 24 hours, ix) ANC of an individual is approximately 0.5 × 10 9 To prevent it from reaching less than / L, x) ANC is approximately 0.5 × 10 9 From the first occurrence of less than / L, approximately 2.0 × 10 within about 10 days 9 Achieve one or more of the following increases: / L or higher. In some embodiments, chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is grade 4 neutropenia.

[0057] In some embodiments, a method is provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring treatment (e.g., a human, e.g., an individual with cancer), wherein the individual is administered at least four cycles (e.g., four cycles) of chemotherapy or radiotherapy (e.g., the chemotherapy or radiotherapy is administered on day 1 of each cycle), and the method is provided for each of the at least four cycles. The method involves administering an effective amount of G-CSF dimer to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2.In some embodiments, methods are provided for treating or preventing a condition characterized by reduced leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring treatment (e.g., a human, e.g., an individual with cancer), wherein the individual is administered at least four cycles (e.g., four cycles) of chemotherapeutic agents or radiotherapy (e.g., the chemotherapeutic agents or radiotherapy are administered on day 1 of each cycle), and the method comprises administering an effective amount of G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agents or radiotherapy in each of the at least four cycles (e.g., 0 hours (co-administration), or 0.5 hours, 3 hours, or 5 hours), wherein the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, methods are provided for treating or preventing a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual requiring treatment (e.g., a human, e.g., an individual with cancer), wherein the individual is administered at least four cycles (e.g., four cycles) of chemotherapy or radiotherapy (e.g., the chemotherapy or radiotherapy is administered on day 1 of each cycle), and the method is performed less than 24 hours after administration of the chemotherapy or radiotherapy in each of the at least four cycles (e.g., The method comprises administering an effective amount of G-CSF dimer to an individual at 0 hours (simultaneous administration), or at 0.5 hours, 3 hours, or 5 hours later, wherein the G-CSF dimer comprises two monomeric subunits, each comprising the amino acid sequence of SEQ ID NO: 6, and the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at a dose of approximately 5 mg to approximately 25 mg per dose, for example, approximately 20 mg per dose. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered a chemotherapeutic agent or radiotherapy for treating cancer (e.g., breast cancer). In some embodiments, the individual is administered two chemotherapeutic agents, docetaxel and cyclophosphamide, for example, simultaneously in each cycle (e.g., in the same formulation or in separate formulations). In some embodiments, the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is approximately 21 days long. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle for at least four cycles. In some embodiments, the administration time of the G-CSF dimer differs in two or more cycles (e.g., 4) of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 5 hours (e.g., 5 hours) after the administration of the chemotherapeutic agent or radiotherapy in each of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 3 hours (e.g., 3 hours) after the administration of the chemotherapeutic agent or radiotherapy in each of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 0.5 hours (e.g., 0.5 hours) after the administration of the chemotherapeutic agent or radiotherapy in each of at least four cycles. In some embodiments, the G-CSF dimer is administered concurrently with (or at 0 hours) the administration of the chemotherapeutic agent or radiotherapy in each of at least four cycles. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.

[0058] In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective dose of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective dose of a G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in each of the at least four cycles (e.g., 0 hours (simultaneous administration), 0.5 hours, 3 hours, or 5 hours). Administration of a G-CSF dimer reduces or prevents a condition characterized by decreased leukocyte production in at least one of four cycles (e.g., cycle 1, or cycles 1-4) (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection), wherein the G-CSF dimer comprises two monomeric subunits, each monomeric subunit comprising a G-CSF monomer (SEQ ID NO: 1), a dimerization domain (e.g., an Fc fragment, e.g., SEQ ID NO: 2), and an optional linker (SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.2.In some embodiments, methods are provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective amount of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective amount of a G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in each of the at least four cycles (e.g., 0 hours (co-administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer mitigates or prevents a condition characterized by decreased leukocyte production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the four or more cycles (e.g., cycle 1, or cycles 1-4), the G-CSF dimer comprising two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., human), the method comprising: i) administering an effective dose of a chemotherapeutic agent or radiotherapy (e.g., the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle) to the individual for at least four cycles (e.g., four cycles); and ii) administering an effective dose of a G-CSF dimer to the individual less than 24 hours after administration of the chemotherapeutic agent or radiotherapy in each of the at least four cycles (e.g., 0 hours (simultaneous administration), or 0.5 hours, 3 hours, or 5 hours later), wherein the administration of the G-CSF dimer is performed for at least four or more cycles. The G-CSF dimer is formulated into a pharmaceutical composition comprising (a) about 20 mg / mL of G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% w / v sorbitol, and (e) about 0.01% w / v polysorbate 20, and the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the G-CSF dimer or pharmaceutical composition is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at a dose of approximately 5 mg to approximately 25 mg per dose, for example, approximately 20 mg per dose. In some embodiments, the condition characterized by decreased leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents, docetaxel and cyclophosphamide, are administered to the individual in each cycle, for example, simultaneously (for example, in the same formulation or in separate formulations).In some embodiments, the chemotherapeutic agent or radiotherapy is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is approximately 21 days long. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of at least four cycles. In some embodiments, the administration time of the G-CSF dimer differs in two or more cycles (e.g., four) of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 5 hours (e.g., 5 hours) after the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 3 hours (e.g., 3 hours) after the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least two cycles, e.g., each cycle of at least four cycles. In some embodiments, the G-CSF dimer is administered within approximately 0.5 hours (e.g., 0.5 hours) after the administration of the chemotherapeutic agent or radiotherapy in each cycle of at least four cycles. In some embodiments, the G-CSF dimer is administered concurrently with (or at 0 hours before) the administration of chemotherapeutic agents or radiotherapy in each of at least four cycles. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.

[0059] In some embodiments, administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of the method herein or a chemotherapeutic agent or radiotherapy induces an increase in the individual's white blood cell (WBC) count (e.g., an increase of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1x, 2x, 5x, 10x, 50x, or more) or a decrease in WBC loss (e.g., a decrease of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). For example, in some embodiments, the neutrophil count (e.g., ANC) increases in the individual (e.g., an increase of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1x, 2x, 5x, 10x, 20x, or more). In some embodiments, the decrease in the neutrophil count is suppressed in the individual (e.g., suppression of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the nadia ANC increases in the individual (e.g., an increase of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1x, 2x, 5x, 10x, or more). In some embodiments, recovered ANC increases in the individual (e.g., an increase of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1x, 2x, 5x, 10x, 20x, or more). In some embodiments, the time to ANC recovery decreases in the individual (e.g., a decrease of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, one or more of the effects described herein are achieved in cycle 1 of chemotherapy or radiotherapy for the individual.In some embodiments, one or more effects described herein are achieved in one or more cycles of chemotherapy or radiotherapy for an individual, for example, cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, one or more effects described herein are achieved in each cycle of chemotherapy or radiotherapy for an individual (for example, all two cycles, or all four cycles).

[0060] In some embodiments, the method described herein, or administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy, reduces the duration of chemotherapy-induced neutropenia (e.g., grade 4) or radiotherapy-induced neutropenia (e.g., grade 4) in the individual (e.g., a reduction of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the duration of chemotherapy-induced neutropenia is reduced by one or more grades of neutropenia, e.g., grade I, grade II, grade III, or grade IV. In some embodiments, the method described herein reduces the duration of grade 4 (severe) neutropenia, i.e., about 0.5 × 10⁻⁶. 9 The duration of ANC less than 1 / L is reduced to less than approximately 24 hours (e.g., less than approximately 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours, or less). In some embodiments, the methods described herein reduce the duration of grade 4 (severe) neutropenia or approximately 0.5 × 10⁻¹⁶ hours. 9 Reduces the duration of ANC below 0.5 × 10 to less than approximately 24 hours. In some embodiments, it is used in grade 4 (severe) neutropenia (ANC < 0.5 × 10). 9 The duration of / L) is when the individual has ANC < 0.5 × 10 in cycle 1 of chemotherapy or radiotherapy. 9This is the number of days having / L. In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia or approximately 0.5 × 10 in cycle 1 of chemotherapy or radiotherapy in an individual. 9 The duration of ANC less than 10 / L is reduced. In some embodiments, the methods described herein reduce the duration of grade 4 neutropenia or reduce the duration of ANC by about 0.5 × 10 in one or more cycles of chemotherapy or radiotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. 9 Reduces the duration of ANC below / L. In some embodiments, the methods described herein reduce the duration of grade 4 neutropenia or reduce the duration of ANC by about 0.5 × 10 in each cycle of chemotherapy or radiotherapy in an individual (e.g., all two cycles or all four cycles). 9 Reduces the duration of ANC below / L. In some embodiments, the methods described herein reduce the duration of grade 3 neutropenia, i.e., about 1.0 × 10⁻¹⁶. 9 The duration of ANC less than 1.0 × 10⁻¹⁰ hours is reduced to less than approximately 24 hours (e.g., less than approximately 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours, or less). In some embodiments, grade 3 neutropenia (ANC < 1.0 × 10⁻¹⁰ hours) is reduced. 9 The duration of / L) is when the individual has ANC < 1.0 × 10 in cycle 1 of chemotherapy or radiotherapy. 9 This is the number of days having / L. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia or approximately 1.0 × 10 in cycle 1 of chemotherapy or radiotherapy in an individual. 9 The duration of ANC less than 1.0 × 10⁻¹⁰ / L is reduced. In some embodiments, the methods described herein reduce the duration of grade 3 neutropenia or reduce the duration of ANC in one or more cycles of chemotherapy or radiotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles by about 1.0 × 10⁻¹⁰ / L.9 Reduces the duration of ANC below / L. In some embodiments, the methods described herein reduce the duration of grade 3 neutropenia or reduce the duration of approximately 1.0 × 10⁻¹⁰ neutropenia in each cycle of chemotherapy or radiotherapy in an individual (e.g., all two cycles or all four cycles). 9 Reduces the duration of ANC below / L.

[0061] In some embodiments, the methods described herein, or administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy, reduces (e.g., reduces by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the incidence of chemotherapy-induced neutropenia (e.g., grade 4) or radiotherapy-induced neutropenia (e.g., grade 4) in the individual, or prevents it. In some embodiments, the duration of chemotherapy-induced neutropenia is reduced by one or more grades of neutropenia, e.g., grade I, grade II, grade III, or grade IV. In some embodiments, the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is reduced or prevented for any grade of neutropenia. In some embodiments, the methods described herein reduce or prevent the incidence of grade 4 (severe) neutropenia. In some embodiments, the methods described herein reduce the ANC in an individual by about 0.5 × 10⁻⁶. 9 To prevent the ANC from reaching less than 0.5 × 10⁻¹⁰. In some embodiments, the methods described herein reduce the incidence of grade 4 neutropenia or the ANC from being about 0.5 × 10⁻¹⁰. 9 The frequency of occurrence reaching less than 10 / L is reduced at least once, for example, at least two, three, four times or more. In some embodiments, the methods described herein reduce or prevent the incidence of grade 4 neutropenia or reduce the ANC of an individual to about 0.5 × 10 in cycle 1 of chemotherapy or radiotherapy. 9The frequency of occurrence reaching less than 10 / L is reduced or prevented. In some embodiments, the methods described herein reduce or prevent the incidence of grade 4 neutropenia or reduce the ANC to about 0.5 × 10 in one or more cycles of chemotherapy or radiotherapy of an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. 9 The frequency of occurrence reaching less than 10 / L is reduced or prevented. In some embodiments, the methods described herein reduce or prevent the incidence of grade 4 neutropenia or reduce the ANC to approximately 0.5 × 10 in each cycle of chemotherapy or radiotherapy of an individual (e.g., all two cycles or all four cycles). 9 It reduces or prevents the incidence of reaching less than 1.0 × 10⁻¹ / L. In some embodiments, the method described herein reduces or prevents the incidence of grade 3 neutropenia. In some embodiments, the method described herein reduces the incidence of ANC in an individual to about 1.0 × 10⁻¹ / L. 9 To prevent the ANC from reaching less than 1.0 × 10⁻¹⁰. In some embodiments, the methods described herein reduce the incidence of grade 3 neutropenia or the ANC from reaching about 1.0 × 10⁻¹⁰. 9 The frequency of occurrence reaching less than 10 / L is reduced at least once, for example, at least two, three, four times or more. In some embodiments, the methods described herein reduce or prevent the incidence of grade 3 neutropenia or the ANC in cycle 1 of chemotherapy or radiotherapy of an individual to about 1.0 × 10 9 The frequency of occurrence reaching less than 1.0 × 10⁻¹⁰ / L is reduced or prevented. In some embodiments, the methods described herein reduce or prevent the incidence of grade 3 neutropenia or reduce the ANC to approximately 1.0 × 10⁻¹⁰ in one or more cycles of chemotherapy or radiotherapy of an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. 9The frequency of occurrence reaching less than 1.0 × 10⁻¹⁰ / L is reduced or prevented. In some embodiments, the methods described herein reduce or prevent the incidence of grade 3 neutropenia or the ANC in each cycle of chemotherapy or radiotherapy of an individual (e.g., all two cycles or all four cycles) 9 Reduce or prevent the frequency of occurrences that reach less than / L.

[0062] In some embodiments, administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of the method herein or a chemotherapeutic agent or radiotherapy reduces or prevents the incidence of febrile neutropenia (FN) in the individual (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). FN is defined as a single oral body temperature of 38.3°C (101°F) or higher, or a body temperature of 38.0°C (100.4°F) or higher for more than one hour, and an ANC of 0.5 × 10⁶ on the same day. 9 ANC is defined as a state of less than / L. Therefore, in some embodiments, the method described herein is defined as a single oral body temperature of 38.3°C (101°F) or higher lasting for more than 1 hour in an individual, or a body temperature of 38.0°C (100.4°F) or higher, and an ANC of 0.5 × 10⁻¹⁰ for the same day (e.g., within approximately 24, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.5 hours or less). 9The method reduces or prevents the incidence of FN below / L. In some embodiments, the method described herein reduces the incidence of FN by at least one, for example, at least two, three, four, or more times. In some embodiments, the method described herein reduces or prevents the incidence of FN in cycle 1 of chemotherapy or radiotherapy for an individual. In some embodiments, the method described herein reduces or prevents the incidence of FN in one or more cycles of chemotherapy or radiotherapy for an individual, for example, cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the method described herein reduces or prevents the incidence of FN in each cycle of chemotherapy or radiotherapy for an individual (for example, all two cycles, or all four cycles).

[0063] In some embodiments, the method described herein, or administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy, reduces the duration and / or incidence of infection in the individual (e.g., reduces by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents its occurrence. In some embodiments, the method described herein reduces the duration and / or incidence of intravenous antibiotic use (e.g., reduces by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the method described herein reduces the frequency of infections by at least one, e.g., at least two, three, four, or more. In some embodiments, the method described herein reduces or prevents the duration and / or incidence of infection in cycle 1 of chemotherapy or radiotherapy in an individual. In some embodiments, the method described herein reduces or prevents the duration and / or incidence of infection in one or more cycles of chemotherapy or radiotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the method described herein reduces or prevents the duration and / or incidence of infection in each cycle of chemotherapy or radiotherapy in an individual (e.g., all two cycles, or all four cycles).

[0064] In some embodiments, the methods described herein, or administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., f-bemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy, reduces the duration and / or incidence of hospitalization due to febrile neutropenia or infection in the individual (e.g., by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents its occurrence. In some embodiments, the methods described herein reduce the duration and / or incidence of intravenous antibiotic use (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the method described herein reduces the incidence of hospitalization for febrile necrosis (FN) or infection by at least one, for example, at least two, three, four, or more times. In some embodiments, the method described herein reduces or prevents the duration and / or frequency of hospitalization for febrile necrosis or infection in cycle 1 of an individual's chemotherapy or radiotherapy. In some embodiments, the method described herein reduces or prevents the duration and / or frequency of hospitalization for febrile necrosis or infection in one or more cycles of an individual's chemotherapy or radiotherapy, for example, cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the method described herein reduces or prevents the duration and / or frequency of hospitalization for febrile necrosis or infection in each cycle of an individual's chemotherapy or radiotherapy (for example, all two cycles, or all four cycles).

[0065] In some embodiments, the method described herein, or administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy, reduces the depth of the individual's ANC nadia (e.g., any reduction of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or increases the nadia value (e.g., any increase of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1x, 2x, 5x, 10x, or more). In some embodiments, the method described herein reduces the depth of the individual's ANC nadia or increases the ANC nadia value in cycle 1 of chemotherapy or radiotherapy. In some embodiments, the method described herein reduces the depth of ANC nadia or increases the ANC nadia value in one or more cycles of chemotherapy or radiotherapy of an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the method described herein reduces the depth of ANC nadia or increases the ANC nadia value in each cycle of chemotherapy or radiotherapy of an individual (e.g., all two cycles, or all four cycles).

[0066] In some embodiments, administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after the administration of the method herein or a chemotherapeutic agent or radiotherapy reduces the time to ANC recovery in the individual (e.g., a reduction of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the time to ANC recovery in the individual is reduced to at least about 5 minutes, 30 minutes, 1 hour, 2 hours, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more. In some embodiments, ANC recovery is defined as the time after an ANC nadia (e.g., expected ANC nadia) when the ANC is 2.0 × 10⁻⁶ 9 The goal is to reach more than / L. Therefore, in some embodiments, the method described herein is used when the ANC Nadia, for example, when the ANC is about 0.5 × 10 9 Since it first became less than / L, ANC is 2.0×10 9 The time to reach 1 / L or more is reduced. In some embodiments, the method described herein, or administration of a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy, reduces the ANC to approximately 0.5 × 10⁻⁶. 9 Within approximately 10 days of first reaching less than / L, for example, within approximately 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less, approximately 2.0 × 10 9 Increase to over / L. In some embodiments, the method described herein results in an ANC of about 0.5 × 10 9 Within approximately 10 days of the first time the level falls below / L, for example, within approximately 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less, approximately 1.5 × 10 9Increase to above / L. In some embodiments, the method described herein reduces the time to ANC recovery in cycle 1 of chemotherapy or radiotherapy for an individual. In some embodiments, the method described herein reduces the time to ANC recovery in one or more cycles of chemotherapy or radiotherapy for an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the method described herein reduces the time to ANC recovery in each cycle of chemotherapy or radiotherapy for an individual (e.g., all two cycles, or all four cycles).

[0067] In some embodiments, one or more effects (e.g., therapeutic effects) achieved by the methods described herein or by administering a G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemalenograstim alfa) to an individual less than 24 hours after administration of a chemotherapeutic agent or radiotherapy are compared with an individual not administered a G-CSF dimer (e.g., any of the G-CSF dimers described herein). In some embodiments, one or more effects achieved by the methods described herein are compared with pegfilgrastim (e.g., Neulasta®) or a biosimilar or efrapegrastim (e.g., Rolontis®, HM10460A, or ROLVEDON®) or a biosimilar, on the same treatment schedule as the G-CSF dimer, i.e., in an individual less than 24 hours after administration of the same chemotherapeutic agent or radiotherapy.

[0068] In some embodiments, the individual is a human. In some embodiments, the G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., fbemalenograstim alfa) or its pharmaceutical composition is administered in approximately 5 mg to approximately 25 mg per dose, for example, approximately 10 to approximately 25 mg, approximately 5 mg to approximately 15 mg, approximately 5 mg to approximately 20 mg, approximately 10 mg to approximately 15 mg, approximately 10 mg to approximately 20 mg, approximately 15 mg to approximately 25 mg, approximately 15 mg to approximately 20 mg, or approximately 18 mg to approximately 22 mg per dose. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered in approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg per dose, or any value in between these. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered at approximately 20 mg per dose. In some embodiments, the subject is a non-human animal. In some embodiments, the G-CSF dimer or its pharmaceutical composition is administered to the non-human animal at a dose equivalent to that of a human. In some embodiments, when the subject is administered two or more cycles of chemotherapy or radiotherapy, the G-CSF dimer or its pharmaceutical composition is administered at the same amount (e.g., 20 mg) in each cycle. In some embodiments, when the subject is administered two or more cycles of chemotherapy or radiotherapy, the G-CSF dimer or its pharmaceutical composition is administered at different amounts in at least two of the cycles.

[0069] The G-CSF dimers (e.g., G-CSF-Fc dimers, e.g., fbemalenograstim alfa) or their pharmaceutical compositions described herein may be administered via any suitable route, including but not limited to intravenous, intra-arterial, intraperitoneal, intravascular, intramuscular, subcutaneous, transmucosal, or transdermal. In some embodiments, sustained-release devices, such as implanted mini osmotic pumps, may be used. Other delivery methods include, but are not limited to, liposomal formulations, intravenous injections, and transdermal patches. In some embodiments, the G-CSF dimers or their pharmaceutical compositions are administered subcutaneously.

[0070] In some embodiments, the individual being treated, or the individual administered a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efbemalenograstim alfa) or its pharmaceutical composition, meets one or more of the following criteria: i) in the case of a human, at least 18 years of age; ii) female if the individual has breast cancer (e.g., stage I-III breast cancer); iii) scheduled to receive two or more cycles (e.g., 2, 3, 4, or more) of chemotherapy or radiotherapy (e.g., neoadjuvant or adjuvant); iv) having an East Coast Cancer Group (ECOG) treatment outcome status of 2 or less; and v) having an ANC ≥ 2.0 × 10 before administration of chemotherapy or radiotherapy. 9 / L, hemoglobin ≥ 11.0 g / dL, and platelet count ≥ 100 × 10 9 vi) having adequate renal, hepatic, and cardiac function (e.g., alanine aminotransferase (ALT), aspartate aminotransferase (AST), and / or alkaline phosphatase are less than 2.5 times the upper limit of normal (ULN); total bilirubin and / or serum creatinine are less than 1.5 × ULN); vii) using contraception at least one month before and during administration of chemotherapeutic agents or radiotherapy. In some embodiments, the methods described herein further include selecting individuals that meet one or more of the above criteria.

[0071] 1. Diseases or conditions induced by chemotherapy agents or radiotherapy The methods described herein, though not limited to these, can be used to treat and / or prevent one or more conditions induced by or associated with chemotherapy and / or radiotherapy, including but not limited to, conditions characterized by a decrease in absolute neutrophil count (ANC) (e.g., a decrease of at least about 5%, 10%, 20%, 50%, 60%, 70%, 80%, 90%, or more), a decrease in granulocyte count, a decrease in stem cell production, a decrease in hematopoiesis, a decrease in hematopoietic progenitor cell (HPC) count, and leukocyte deficiency.

[0072] In some embodiments, conditions characterized by decreased leukocyte production include one or more of the following: chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, decreased hematopoietic function, decreased immune function, decreased neutrophil count, decreased neutrophil recruitment, mobilization of peripheral blood progenitor cells, sepsis, infection, leukopenia, low bone marrow engraftment during transplantation, decreased bone marrow recovery in the treatment of myeloplasty or myelosuppression induced by radiation, chemicals or chemotherapy, radiotherapy-induced myeloplasty or myelosuppression, and acquired immunodeficiency syndrome. In some embodiments, conditions characterized by decreased leukocyte production are chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.

[0073] Neutropenia Neutropenia is a condition in which the number of neutrophils in the peripheral blood is 1.8 × 10⁻⁶ in adults. 9 Less than / L, 1.5 × 10 in children 9 It is characterized by a neutropenia of less than / L. Neutropenia is often a precursor to infection, and the lower the neutrophil count, the higher the risk of infection.

[0074] Table A shows the guidelines used to classify neutropenia. The frequency and severity of infections caused by neutropenia are also influenced by other factors such as mucosal and skin integrity, immunoglobulins, lymphocytes, monocytes, and complement system function and levels. [Table 1]

[0075] Depending on the cause of neutropenia, general clinical neutropenia can be classified into the following categories: impaired hematopoietic production caused by secondary factors such as drugs, radiation, chemicals, and infections; in vivo changes in distribution and circulation, increased utilization, and increased turnover. The severity of chemotherapy-induced neutropenia in cancer patients generally depends on the dose of chemotherapy, and repeated use of chemotherapy can have a cumulative effect on neutropenia. The main clinical outcome of neutropenia is the complication of infection. Many of the infections in these patients are mainly caused by aerobic bacteria, including, for example, Gram-negative bacteria (Escherichia coli, Klebsiella pheumoniae, and Pseudomonas aeruginosa), Gram-positive bacteria (Staphylococci, α-hemolytic Streptococci, and Straphylococcus aureus), as well as fungi.

[0076] Cytotoxic chemotherapy remains one of the primary treatments for cancer. The biggest drawback of chemotherapy is that it indiscriminately kills healthy cells that rapidly proliferate and differentiate, even if they are tumor cells. The toxicity caused by chemotherapy is primarily reflected in the hematopoietic system and is clinically known as chemotherapy-induced neutropenia.

[0077] Neutropenia can delay the next treatment cycle and directly affect the effectiveness of chemotherapy. Severe neutropenia, i.e., ANC of 0.5 × 10⁻⁶ 9 Levels below / L can cause infection and organ failure in patients, and may be life-threatening.

[0078] In some embodiments, neutropenia is grade 1 neutropenia. In some embodiments, neutropenia is grade 2 neutropenia. In some embodiments, neutropenia is grade 3 neutropenia. In some embodiments, neutropenia is grade 4 neutropenia or severe neutropenia. In some embodiments, neutropenia is febrile neutropenia (FN).

[0079] Accordingly, in some embodiments, methods are also provided for treating or preventing neutropenia in individuals in need (e.g., humans, e.g., individuals with cancer), the methods comprising administering an effective amount of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efbemarenograstim alfa) less than 24 hours (e.g., 0 hours (simultaneous with administration), or 0.5 hours, 3 hours, or 5 hours later) after administering a neutropenia-inducing agent, e.g., a neutropenia-inducing chemotherapeutic agent or radiotherapy to the individual.

[0080] Diseases treated with chemotherapy or radiation therapy In some embodiments, the individual is administered a chemotherapy agent or radiotherapy to treat cancer.

[0081] Cancers that can be treated include, but are not limited to, colorectal cancer, breast cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, melanoma, liver cancer, head and neck cancer, glioma, gallbladder cancer, pancreatic cancer, prostate cancer, duodenal papilla cancer, esophageal cancer, kidney cancer, thyroid cancer, squamous cell carcinoma, lung cancer, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), Burkitt lymphoma, and Wilms' tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor.

[0082] In some embodiments, the cancer is a non-myeloid cancer, such as a non-myeloid malignant tumor. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is stage I-III or stage II-IV breast cancer.

[0083] In some embodiments, the method of administering the G-CSF dimer described herein ensures that the individual does not delay the treatment schedule of the chemotherapy agent or radiotherapy, or that any delay in the treatment schedule of the chemotherapy agent or radiotherapy is within about 5 days, for example, about 4.5, 4, 3.5, 3, 2.5, 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.6, 0.4, 0.2, 0.1 days, or less. In some embodiments, when two or more cycles of chemotherapy agent or radiotherapy are administered, the method of administering the G-CSF dimer described herein ensures that the individual does not delay the treatment schedule of the chemotherapy agent or radiotherapy within each cycle, or that the delay is only in cycle 2 (e.g., about 5 days or less), or that there is no delay in any cycle (e.g., only cycles 2 and 3 of four cycles).

[0084] Chemotherapy agents In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.

[0085] In some embodiments, the cancer treated by the chemotherapy agent is breast cancer. In some embodiments, the individual is given i) epirubicin and cyclophosphamide (e.g., epirubicin 100 mg / m²). 2 and cyclophosphamide 600 mg / m² 2 ), ii) Docetaxel and cyclophosphamide (e.g., docetaxel 75 mg / m²) 2 and cyclophosphamide 600 mg / m² 2 iii) doxorubicin and cyclophosphamide, iv) docetaxel and docetaxel (e.g., docetaxel 75 mg / m²) 2 and doxorubicin 60 mg / m² 2), v) docetaxel, doxorubicin and cyclophosphamide, or vi) cyclophosphamide, doxorubicin and vincristine (e.g., cyclophosphamide 750 mg / m²) 2 , Doxorubicin 50 mg / m² 2 , and vincristine 1.4 mg / m² 2 Two or more chemotherapeutic agents (up to 2.0 mg) are administered. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially.

[0086] In some embodiments, the chemotherapeutic agent induces neutropenia, such as grade 4 neutropenia (e.g., neutropenia of any grade). In some embodiments, the chemotherapeutic agent induces febrile nephropenia (FN). In some embodiments, the chemotherapeutic agent increases ANC by approximately 0.5 × 10⁻⁶. 9 Reduce to less than / L. In some embodiments, the chemotherapeutic agent reduces ANC, for example, by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of ANC. In some embodiments, the chemotherapeutic agent induces a duration (DN) of neutropenia of at least about 2 days, for example, at least about 3, 4, 5, 6, 7, 8, 9, or 10 days or longer.

[0087] Radiation therapy Radiation therapy, also known as radiotherapy or therapeutic radiology, is the use of radioactive sources in the treatment or palliative care of a disease. Radiation therapy typically utilizes ionizing radiation and deep-tissue penetrating light, which can physically and chemically react with and destroy affected cells. Each therapy program has a radiation dose defined by the type and amount of radiation in each treatment session, the frequency of treatment sessions, and the total number of sessions.

[0088] Radiotherapy is particularly well-suited for treating solid tumors with a clear spatial contour. Such tumors are found in breast cancer, kidney cancer, and prostate cancer, as well as in secondary proliferations of the brain, lungs, and liver.

[0089] Traditionally, the mainstream of radiotherapy has been so-called external beam radiation therapy, which involves treating tumors growing within a human body using radiation from an external source (e.g., gamma rays). Alternatively, a radiation source (usually an electron emitter) is inserted into the body.

[0090] To avoid adverse effects on any healthy areas of the target, attempts are made to maximize the dose delivered to the target area (ensuring the death of cancer cells) while minimizing the dose to other areas (to avoid undesirable damage). Radiotherapy is most commonly used as an adjunct treatment, for example, to treat residual tumor cells that were not completely removed by irradiating them with radiation from an external source after surgically opening the body to remove a malignant tumor and then suturing the body part, or by directly irradiating residual tumor cells with radiation before suturing the body part.

[0091] The efficiency of cell killing varies greatly depending on the type of radiation. Gamma rays and beta rays are relatively inefficient. In contrast, alpha particles and other heavy charged particles can deliver more energy and are therefore very efficient. Under certain conditions, the energy delivered by a single heavy particle is sufficient to destroy a cell. Furthermore, because heavy particles can deliver radiation over distances of several cell diameters, nonspecific irradiation of normal tissue surrounding the target cell is greatly reduced or absent.

[0092] In some embodiments, the radiation therapy is myelosuppressive. In some embodiments, the radiation therapy induces neutropenia, such as grade 4 neutropenia (e.g., neutropenia of any grade). In some embodiments, the radiation therapy induces FN. In some embodiments, the radiation therapy reduces the ANC to less than about 0.5 × 10 9 / L. In some embodiments, the radiation therapy reduces the ANC, e.g., reduces the ANC by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%. In some embodiments, the radiation therapy induces a duration of neutropenia (DN) of at least about 2 days, e.g., at least about 3, 4, 5, 6, 7, 8, 9, 10 days, or more.

[0093] 2. G-CSF Human granulocyte colony-stimulating factor (G-CSF) is a glycoprotein having 204 amino acids, including a 30-amino acid signal peptide. The mature G-CSF protein has a molecular weight of 18-20 kDa and is composed of 174 amino acids without the signal peptide and is secreted extracellularly. The human cells mainly responsible for such secretion are monocytes, fibroblasts, and endothelial cells. G-CSF has three main biological functions: 1) acting on bone marrow precursors and stem cells to induce neutrophil differentiation, development, and maturation; 2) activating mature neutrophils to participate in the immune response; and 3) acting together with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand, and GM-CSF to mobilize hematopoietic stem cells.

[0094] The G-CSF receptor (G-CSFR) has been shown to be present in bone marrow hematopoietic stem cells (Sca+Lin-Th1low), precursor cells (CD34+), committed granulocyte precursor cells, and mature neutrophils. G-CSFR is a specific receptor with high affinity for G-CSF and consists of 812 amino acids. Tamada et al. obtained the crystal structure of the G-CSF:G-CSFR complex and showed the stoichiometry of the G-CSF:G-CSFR complex as a 2:2 ratio by diffraction analysis at 2.8 angstroms (PNAS, 2008, Vol.103:3135-3140). In other words, in each complex, each G-CSF binds to one receptor chain to form a G-CSF-receptor complex, and when two G-CSF-receptor complexes are in close proximity, a 2:2 dimer is formed as a result of this interaction. Under these conditions, the carboxyl terminus of the G-CSF receptor can activate the downstream signaling molecule JAK (Janus tyrosine kinase, mainly JAK2). As a result, JAK2 activates STAT3, which in turn switches on transcription genes crucial for neutrophil differentiation, proliferation, and activation.

[0095] In 2003, Schhabitz et al. reported that rhG-CSF had a protective function against nerve cells in an ischemic animal model (Storke, 2003, 34; 745-751). Subsequently, in 2006, Schu et al. reported that rhG-CSF had a clinical effect in the treatment of patients with acute stroke who received daily rhG-CSF for five consecutive days (CMAJ, 2006, 174: 927-933). The in vivo half-life of subcutaneously administered rat G-CSF is approximately 2 hours, while the half-life of subcutaneously administered human G-CSF is only 3.5 hours. Therefore, patients requiring treatment need to be administered the drug daily or intravenously, which affects the patient's quality of life.

[0096] Pegfilgrastim, also known as Neulasta®, SD-01, and PEG-rmetHuG-CSF, is a G-CSF drug developed to reduce the severity and duration of severe neutropenia, as well as neutropenic complications associated with the administration of myelosuppressive anticancer drugs or radiotherapy. Pegfilgrastim consists of recombinant methionyl human G-CSF (r-metHuG-CSF) covalently bonded to monomethoxypolyethylene glycol (mPEG) via an amide bond. r-metHuG-CSF is identical to natural human G-CSF (SEQ ID NO: 1), except for the N-terminal methionine required for expression in E. coli. The binding of mPEG to the N-terminal methionine of r-metHuG-CSF is thought to extend the serum half-life of r-metHuG-CSF (approximately 15-80 hours), thereby reducing the frequency of administrations required to maintain therapeutically effective neutrophil counts.

[0097] ROLONTIS®, SPI-2012, HM10460A, and 17,65 Efrapegrastim, also known as SG-CSF, is a long-acting G-CSF drug developed to reduce the severity and duration of severe neutropenia, as well as neutropenia complications associated with the use of myelosuppressive anticancer drugs or radiotherapy. Efrapegrastim consists of a recombinant human G-CSF analog (ef-G-CSF) linked by a bifunctional polyethylene glycol linker and a recombinant fragment of the Fc region of human IgG4. ef-G-CSF differs from human G-CSF (SEQ ID NO: 1) at positions 17 and 65, which are substituted with serine. The Fc region of human IgG4 is thought to increase the serum half-life of ef-G-CSF to approximately 36.4 hours (ranging from approximately 16.1 to 115 hours).

[0098] Both pegfilgrastim and efrapegrastim contain monomeric G-CSF molecules.

[0099] 3.G-CSF dimer Any of the G-CSF dimers described herein can be used in the method of the present invention.

[0100] As used herein, the term “G-CSF dimer” refers to a protein comprising (or essentially comprising) two units of a G-CSF molecule, for example, two units of any of the G-CSF monomers described herein, or two units of a monomeric subunit comprising any of the G-CSF monomers described herein. In an unrestricted example, a G-CSF dimer may comprise (or essentially comprise, or may comprise) two G-CSF monomers directly linked to each other or linked together via a linking portion such as a peptide linker, chemical bond, covalent bond, or polypeptide (e.g., a carrier protein, dimerizing domain). In another unrestricted example, a G-CSF dimer may comprise two monomeric subunits, each comprising a G-CSF monomer linked to a carrier protein (e.g., albumin, or Fc domain). Further examples of G-CSF dimers that can be used in the present invention are described in U.S. Patent No. US8557546, U.S. Patent No. US9642917, U.S. Patent Application No. US20130165637, and PCT / CN2022 / 113560 filed on 19 August 2022, the entire contents of which are incorporated herein by reference.

[0101] As used herein, the term “G-CSF monomer” refers to a single unit of the G-CSF protein or molecule. The terms “G-CSF,” “G-CSF molecule,” and “G-CSF protein” are used interchangeably herein.

[0102] The G-CSF monomer used in the G-CSF dimer can be derived from any organism, including, for example, humans, or non-human animals, including mammals such as primates (e.g., crab-eating macaques, rhesus macaques), cattle, pigs, horses, sheep, goats, rodents, cats, and dogs, but not limited to these. The G-CSF monomer may be wild-type G-CSF or mutant G-CSF, for example, mutant G-CSF that can produce most or all the bioactivity of wild-type G-CSF. In some embodiments, the G-CSF monomer is mature G-CSF. In some embodiments, the G-CSF monomer is a functional fragment of G-CSF that can produce most or all the bioactivity of full-length or mature G-CSF. In some embodiments, the G-CSF monomer is mouse G-CSF. In some embodiments, the G-CSF monomer is human G-CSF (hG-CSF). In some embodiments, the G-CSF monomer contains (or is derived from) the amino acid sequence of SEQ ID NO: 1.

[0103] In some embodiments, the G-CSF dimer is a recombinant protein containing two G-CSF (e.g., hG-CSF) molecules produced, for example, within a suitable host cell (e.g., a CHO cell). In some embodiments, the G-CSF dimer contains (or is essentially derived from) two G-CSF (e.g., hG-CSF) monomers, such as two G-CSF monomers linked to each other via a linker (e.g., a peptide linker). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are the same (e.g., both contain the sequence of Sequence ID No. 1). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are different.

[0104] In some embodiments, a G-CSF dimer comprises (or is essentially composed of, or consists of) two G-CSF (e.g., hG-CSF) monomers and a carrier protein. In some embodiments, the carrier protein is albumin (e.g., human albumin). In some embodiments, the carrier protein is the Fc domain of an immunoglobulin (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are the same (e.g., both contain the sequence of Sequence ID No. 1). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are different. In some embodiments, the G-CSF dimer comprises formula (I): M1-L-M2(I) (wherein M1 is a first monomer of G-CSF, M2 is a second monomer of G-CSF, and L is a linker positioned between the first and second monomers, connecting them.) In some embodiments, the linker L is selected from the group consisting of i) short-chain peptides containing about 3 to about 50 amino acids, and ii) polypeptides of formula (II): -ZYZ-(II) (wherein Y is a carrier protein (e.g., albumin, Fc domain), Z is null or a short-chain peptide (or more) containing about 1 to about 30 amino acids, and "-" indicates a chemical or covalent bond). This specification may use any suitable linker or short peptide that can provide flexibility between two G-CSF monomers or between a G-CSF monomer and a carrier protein, and / or ensure the binding of each G-CSF monomer to its receptor. In some embodiments, the linker L or short-chain peptide Z comprises any of the amino acid sequences of SEQ ID NOs: 12-31.

[0105] The carrier proteins described herein may be any protein suitable for linking two G-CSF monomers to form a G-CSF dimer, including, but not limited to, immunoglobulin Fc fragments (e.g., human IgG1, IgG2, IgG3, IgG4) or albumin (e.g., human serum albumin). When a carrier protein is formed by linking two protein subunits (e.g., via disulfide bonds, peptide bonds, or chemical bonds), each protein subunit is called a dimerizing domain. In some embodiments, the carrier protein is formed by linking two dimerizing domains (e.g., two Fc fragments of IgG) via one or more disulfide bonds. In some embodiments, the two dimerizing domains forming the carrier protein are the same (e.g., two IgG2Fc fragments). In some embodiments, the two dimerizing domains forming the carrier protein are different. For example, in some embodiments, the carrier protein is formed by linking a first Fc fragment to a second different Fc fragment via one or more disulfide bonds.

[0106] In some embodiments, a G-CSF dimer comprises (or is essentially composed of or consists of) two monomeric subunits, each monomeric subunit comprising (or is essentially composed of or consists of) a G-CSF monomer (e.g., an hG-CSF monomer) and a dimerization domain. If the dimerization domain is an Fc fragment, such a G-CSF dimer is also called a "G-CSF-Fc dimer".

[0107] In some embodiments, the G-CSF monomer includes the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least any of these). In some embodiments, the G-CSF monomer includes the amino acid sequence of SEQ ID NO: 1.

[0108] In some embodiments, within each monomer subunit, the G-CSF monomer is directly attached to the dimerization domain.

[0109] In some embodiments, within each monomeric subunit, the G-CSF monomer is connected to the dimerization domain via an optional linker. In some embodiments, the linker has a length of about 6 to about 30 amino acids, for example, any length of about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the linker is 16 amino acids long. Any suitable linker that can provide flexibility between the G-CSF monomer and the dimerization domain can be used herein. In some embodiments, the linker includes any amino acid sequence of SEQ ID NOs: 12 to 31. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 12.

[0110] The linker located between two G-CSF monomers, or between one G-CSF monomer and a carrier protein or dimerization domain, may be null (no linker), a peptide linker, or a non-peptide linker. In some embodiments, the first linker connecting the first G-CSF monomer to the carrier protein (or the first dimerization domain) is the same as the second linker connecting the second G-CSF monomer to the carrier protein (or the second dimerization domain). In some embodiments, the first linker connecting the first G-CSF monomer to the carrier protein (or the first dimerization domain) is different from the second linker connecting the second G-CSF monomer to the carrier protein (or the second dimerization domain). Generally, the linker does not affect, or significantly affects, the proper folding and conformation formed by the stereochemistry of the two G-CSF monomers.

[0111] The linker may be a peptide linker of any length. In some embodiments, the peptide linker is about 1 amino acid (aa) to about 10aa in length, about 2aa to about 15aa in length, about 5aa to about 8aa in length, about 1aa to about 20aa in length, about 21aa to about 30aa in length, about 1aa to about 30aa in length, about 10aa to about 30aa in length, about 3aa to about 50aa in length, or about 6aa to about 30aa in length. In some embodiments, the peptide linker is any length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the peptide linker is any length of about 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the peptide linker is of any length of approximately 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. For example, in some embodiments, the linker is of a length of 3 to approximately 50 amino acids. In some embodiments, the linker is of a length of approximately 6 to approximately 30 amino acids.

[0112] Peptide linkers may have naturally occurring or non-naturally occurring sequences. For example, sequences derived from the hinge region of heavy-chain-only antibodies can be used as linkers. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is the hinge of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the peptide linker contains the amino acid sequence of SEQ ID NO: 13. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G). n (SEQ ID NO: 18), Glycine-serine polymer (e.g., (GS) n (Sequence ID 19), (GSGGS) n (Sequence ID 20), or (GGGGS) nExamples include (including SEQ ID NO: 22) (where n is an integer at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Because glycine polymers and glycine-serine polymers are relatively unstructured, they can function as neutral tethers between components. Glycine's access in the fi-psy space is significantly greater than that of alanine and far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Exemplary flexible linkers include, but are not limited to, any of SEQ ID NOs: 12 and 14-31. In some embodiments, the linker includes (or is essentially derived from, or becomes derived from) the amino acid sequence of SEQ ID NO: 12. Those skilled in the art will recognize that the design of a G-CSF dimer may include a linker that is entirely or partially flexible, so that the linker includes not only a flexible linker portion but also one or more portions that provide a less flexible structure in giving the desired G-CSF dimer structure and function.

[0113] In some embodiments, the linker between a G-CSF monomer and a carrier protein (e.g., a dimerization domain), or between two G-CSF monomers, is a stable linker (not cleaved by proteases, particularly MMPs).

[0114] Other linker considerations include their influence on the physical and pharmacokinetic properties of the resulting G-CSF dimer, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (some degree of stability and planned degradation), rigidity, flexibility, immunogenicity, modulation of G-CSF / G-CSF receptor binding, and the ability to be incorporated into micelles or liposomes.

[0115] In some embodiments, the dimerization domain (e.g., Fc fragment) within each monomeric subunit contains at least two cysteine ​​molecules capable of forming intermolecular disulfide bonds. In some embodiments, there are about 2 to about 4 disulfide bonds between two dimerization domains (e.g., Fc fragments). In some embodiments, the dimerization domain contains a leucine zipper. In some embodiments, the dimerization domain contains at least a portion of the Fc fragment. In some embodiments, the Fc fragment contains CH2 and CH3 domains. In some embodiments, the dimerization domain is derived from an Fc fragment of IgA, IgD, IgE, IgG, and IgM, or any of their subtypes. In some embodiments, the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or a fragment or variant thereof. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG2. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG1. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG4. In some embodiments, the dimerization domain is a wild-type Fc fragment. In some embodiments, the dimerization domain includes one or more mutations in the Fc fragment that reduce or eliminate effector function (e.g., reducing antibody-dependent cell-mediated cytotoxicity (ADCC) or reducing binding to FcγR). In some embodiments, the Fc fragment includes L234A and L235A ("LALA") substitutions (EU numbering). In some embodiments, the dimerization domain is an IgG2 Fc fragment including a P331S substitution by EU numbering. In some embodiments, the IgG2 Fc fragment includes the sequence of SEQ ID NO: 2 or 3. In some embodiments, the dimerization domain is an IgG1 Fc fragment including L234A, L235A, and P331S substitutions by EU numbering. In some embodiments, the IgG1 Fc fragment includes the sequence of SEQ ID NO: 4. In some embodiments, the dimerization domain is an IgG4 Fc fragment containing the EU numbering S228P, L234A, and L235A substitutions. In some embodiments, the IgG4 Fc fragment contains the sequence of Sequence ID No. 5.In some embodiments, the dimerization domain includes a full-length Fc fragment. In some embodiments, the dimerization domain includes an N-terminal shortened Fc fragment, for example, a shortened Fc fragment with fewer N-terminal cysteine ​​to reduce mispairing of disulfide bonds during dimerization. In some embodiments, the Fc fragment is shortened at the N-terminus, for example, lacking the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the complete immunoglobulin Fc domain. In some embodiments, the dimerization domain is an IgG2 Fc fragment from which the N-terminal "ERKCC" sequence (SEQ ID NO: 32) has been removed.

[0116] In some embodiments, each monomer subunit comprises an Fc fragment of human IgG2 or a variant thereof containing up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each monomer subunit comprises an Fc fragment of human IgG1 or a variant thereof containing up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each monomer subunit comprises an Fc fragment of human IgG4 or a variant thereof containing up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each monomer subunit comprises an Fc fragment having at least 95% sequence identity to any of SEQ ID NOs: 2-5. In some embodiments, the Fc fragment comprises (or is essentially derived from, or becomes derived from) the sequences of SEQ ID NOs: 2-5. In some embodiments, the Fc fragment comprises (or is essentially derived from, or becomes derived from) the amino acid sequence of SEQ ID NO: 2.

[0117] In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein i) each monomeric subunit comprises a G-CSF monomer containing the amino acid sequence of SEQ ID NO: 1 and an Fc fragment containing the amino acid sequence of SEQ ID NO: 2; ii) each monomeric subunit comprises a G-CSF monomer containing the amino acid sequence of SEQ ID NO: 1 and an Fc fragment containing the amino acid sequence of SEQ ID NO: 3; iii) each monomeric subunit comprises a G-CSF monomer containing the amino acid sequence of SEQ ID NO: 1 and an Fc fragment containing the amino acid sequence of SEQ ID NO: 4; or iv) each monomeric subunit comprises a G-CSF monomer containing the amino acid sequence of SEQ ID NO: 1 and an Fc fragment containing the amino acid sequence of SEQ ID NO: 5. In some embodiments, within each monomer subunit, the G-CSF monomer is linked to an Fc fragment via a linker, such as a linker containing the amino acid sequence of SEQ ID NO: 12.

[0118] In some embodiments, the G-CSF monomer is located at the C-terminal end of the dimerization domain within each monomer subunit. In some embodiments, the G-CSF monomer is located at the N-terminal end of the dimerization domain within each monomer subunit.

[0119] In some embodiments, each monomeric subunit includes the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 6 (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, each monomeric subunit includes the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 7 (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, each monomeric subunit includes the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 8 (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any of these). In some embodiments, each monomeric subunit includes the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 9 (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any of these). In some embodiments, each monomeric subunit includes the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0120] In some embodiments, each monomeric subunit contains one of the amino acid sequences of SEQ ID NOs: 6 to 10. In some embodiments, each monomeric subunit contains the amino acid sequence of SEQ ID NO: 6.

[0121] Therefore, in some embodiments, the G-CSF dimer comprises two monomeric subunits, each containing the amino acid sequence of SEQ ID NO: 6. This G-CSF dimer (or G-CSF-Fc dimer) is also referred to herein as "F-627" or "F-bemalenograstim alpha". In some embodiments, each monomeric subunit of the G-CSF dimer is encoded by a nucleic acid containing the sequence of SEQ ID NO: 11.

[0122] In some embodiments, amino acid sequences that do not affect the biological activity of the G-CSF dimer can be added to the N-terminus or C-terminus of one or both monomer subunits of the G-CSF dimer (e.g., the G-CSF monomer or the G-CSF-Fc monomer subunit). In some embodiments, such added amino acid sequences are effective for expression (e.g., signal peptides), purification (e.g., 6×His sequences, cleavage sites of the Saccharomyces cerevisiae α-factor signal peptide (Glu-Lys-Arg)), or enhancement of the biological activity of the G-CSF dimer.

[0123] In some embodiments, the G-CSF dimer is ephemalenograstim alfa. Ephemalenograstim alfa is a long-acting fusion protein of human G-CSF and human IgG2 Fc fragments, existing as a dimer consisting of two G-CSF-Fc monomers covalently linked via a disulfide bond formed between the Fc fragments of the molecule. Each G-CSF-Fc monomer submit contains the amino acid sequence of SEQ ID NO: 6. Each hIgG2 Fc monomer subunit has a P297S substitution when numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomer subunit, which corresponds to a P331S substitution located in the CH2 domain according to EU numbering. The purpose of the P331S substitution is to reduce complement-dependent cytotoxicity (CDC) mediated by the Fc region of human IgG2. Ephemalenograstim alfa is a glycosylated protein produced by Chinese hamster ovary cells using serum-free medium. Each G-CSF-Fc monomer subunit of f-bemalenograstim alfa has one N-linked glycosylation site at N263 when the entire polypeptide chain of the G-CSF-Fc monomer subunit is numbered from the N-terminus, which corresponds to N297 located in the CH2 domain of the Fc region according to EU numbering. Each G-CSF-Fc monomer subunit also has one O-linked glycosylation site at T133 of the human G-CSF monomer. While we do not wish to be bound by theory, it is thought that the IgG2 Fc fragment extends the serum half-life of human G-CSF.

[0124] Through specific binding to the G-CSF receptor, efebmalenograstim alfa stimulates the survival, proliferation, differentiation, and function of neutrophil progenitor cells and mature neutrophils. Efebmalenograstim alfa was previously developed to reduce the incidence of infections presenting as febrile neutropenia in patients with non-myeloid malignancies treated with myelosuppressive anticancer drugs in clinically significant cases of febrile neutropenia (efebmalenograstim alfa is administered after myelosuppressive anticancer drugs, e.g., 24 or 48 hours later).For example, clinical trials NCT03252431 and NCT02872103: Glaspy et al., “A Phase III, Randomized Multi-Center, Open-Label, Fixed-Dose, Neulasta Active-Controlled Clinical Trial of F-627, a Novel G-CSF, in Women with Breast Cancer Receiving Myelotoxic Chemotherapy,” Blood (2021) 138 (Supplement 1):4290; Daley et al., “Abstract P5-16-14: A randomized, multicenter phase III study of once-per-cycle administration of efbemalenograstim alfa(F-627), a novel long-acting dimeric rhG-CSF, for prophylaxis of chemotherapy-induced neutropenia in patients with breast cancer,” Cancer Res (2022) 82(4_Supplement):P5-16-14; Glaspy et al. See al., “A phase III, randomized, multi-centre, double-blind, placebo-controlled clinical trial of F-627 in women with breast cancer receiving myelotoxic chemotherapy,” 2018 San Antonio Breast Cancer Symposium, Publication Number: P6-13-03.

[0125] (a) Substitutions, insertions, deletions, and variants In some embodiments, G-CSF dimers having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table B under the heading "Preferred Substitutions." More substantial changes are shown in Table B under the heading "Exemplary Substitutions," and with respect to amino acid side chain classes, are further described below. Amino acid substitutions can be introduced into G-CSF dimers (e.g., substitutions in one or more dimerizing domains such as the G-CSF monomer, linker, or Fc fragment), and the product can be screened for desired activity, e.g., retention / improvement of receptor binding, reduction of immunogenicity, or improvement of ADCC or complement-dependent cell-mediated cytotoxicity (CDC). [Table 2]

[0126] Amino acids can be classified into groups based on common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0127] Non-conservative substitution involves swapping a member of one class with a member of another class.

[0128] Modifications (e.g., substitutions) can be made to improve, for example, G-CSF affinity.

[0129] In some embodiments, substitutions, insertions, or deletions may occur within the G-CSF dimer, provided that such modifications do not significantly reduce the G-CSF dimer's ability to bind to the G-CSF receptor. For example, conservative modifications that do not significantly reduce binding affinity (e.g., conservative substitutions provided herein) can be made.

[0130] (b) Glycosylated variants In some embodiments, each G-CSF-Fc monomer subunit within the G-CSF dimer has one N-linked glycosylation site at N297, located in the CH2 domain according to EU numbering. In some embodiments, each G-CSF-Fc monomer subunit within the G-CSF dimer has one O-linked glycosylation site at T133 of the hG-CSF monomer relative to Sequence ID No. 1.

[0131] In some embodiments, the G-CSF dimer is modified to increase or decrease the degree to which the protein is glycosylated. Addition or removal of glycosylation sites (e.g., to Fc) can be easily carried out by modifying the amino acid sequence so that one or more glycosylation sites are formed or removed.

[0132] When a G-CSF dimer contains an Fc region, it can modify the carbohydrate bound to it. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides, which generally have an Fc region. H It is N-linked to N297 of the two domains. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to GlcNAc in the "stem" of a branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the Fc region can be performed to create Fc variants with improved specific properties.

[0133] In some embodiments, the G-CSF-Fc dimer has a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc domain. For example, the amount of fucose in such a G-CSF-Fc dimer may be about 1% to about 80%, about 1% to about 65%, about 5% to about 65%, or about 20% to about 40%. As described in WO2008 / 077546, for example, the amount of fucose is determined by calculating the average amount of fucose at N297 in the glycan relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to N297 as measured by MALDI-TOF mass spectrometry. N297 refers to an asparagine residue located around position 297 in the Fc domain (in the EU numbering of Fc domain residues), although N297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to slight sequence changes in the Fc domain. Such fucose-based variants may have improved ADCC functionality. See, for example, U.S. Patent Application Publication No. US2003 / 0157108 (Presta, L.); U.S.2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "defucosed" or "fucose-deficient" proteins include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, and Okazaki et al. References include al. J. Mol. Biol. 336:1239-1249 (2004) and Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated proteins include Lec13CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., particularly Example 11), and knockout cell lines such as CHO cells with the alpha-1,6-fucosyltransferase gene FUT8 knocked out (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0134] In some embodiments, the G-CSF-Fc dimer has a bifid oligosaccharide, for example, a branched oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such variants may have reduced and / or improved ADCC function due to fucosylation.

[0135] (c) Fc domain variant In some embodiments, the Fc fragment of the G-CSF-Fc dimer has reduced effector function compared to the corresponding wild-type Fc domain (e.g., a reduction of at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% as measured by the level of antibody-dependent cytotoxicity (ADCC)).

[0136] In some embodiments, Fc region variants can be created by introducing one or more amino acid modifications into the Fc region of a G-CSF-Fc dimer. Fc region variants may include human Fc region sequences containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0137] In some embodiments, Fc fragments possess some, but not all, effector functions, making them desirable candidates in applications where certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental, although the half-life of the G-CSF-Fc dimer in vivo is important. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to determine that the G-CSF-Fc dimer lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. Natural killer (NK) cells, the main cells mediating γADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells.Alternatively, or in addition to the above, the ADCC activity of the target molecule can be evaluated in vivo using animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Alternatively, a C1q binding assay can be performed to confirm that the G-CSF-Fc dimer cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods well known in the art (see, for example, Petkova et al., Int'l.Immunol. 18(12):1759-1769 (2006)).

[0138] Fc domains with reduced effector function include those having one or more substitutions among Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (EU numbering). Such Fc mutants include the so-called "DANA" Fc mutant having substitutions at residues 265 and 297 to alanine (U.S. Patent No. 7,332,581), and Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327. In some embodiments, the Fc fragment includes the N297A mutation. In some embodiments, the Fc fragment includes the N297G mutation.

[0139] Specific Fc variants with improved or reduced binding to FcR are also described (see, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0140] In some embodiments, modifications are made to the Fc region that result in alterations (either improvement or reduction) of C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0141] In some embodiments, the G-CSF-Fc dimer includes a variant Fc region containing one or more amino acid substitutions that alter the half-life and / or the binding to the fetal Fc receptor (FcRn). Fc domains with a longer half-life and improved binding to the fetal Fc receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus, are described in US2005 / 0014934A1 (Hinton et al.). For example, these Fc domains with one or more substitutions, such as a substitution at Fc domain residue 434, have altered binding affinity of the Fc region to FcRn (U.S. Patent No. 7,371,826).

[0142] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260; U.S. Patent Nos. 5,624,821; and WO94 / 29351.

[0143] In some embodiments, it may be desirable to prepare cysteine-modified G-CSF dimers in which one or more residues of the G-CSF dimer are substituted with cysteine ​​residues. Modified G-CSF dimer variants can provide a variety of intermolecular and intramolecular bonds.

[0144] In some embodiments, the C-terminal lysine (K447) of the Fc region can be cleaved by endogenous carboxypeptidase digestion in mammalian cell culture. The absence of C-terminal lysine does not affect the structure or stability of the Fc region. See Harris et al., Journal of Chromatography A, 705 (1995) 129-134. In some embodiments, the G-CSF dimer contains an Fc region with the C-terminal lysine residue cleaved. In some embodiments, in a formulation of a mixture of G-CSF dimers containing the Fc region, at least some of the G-CSF dimers contain an Fc region with the C-terminal lysine residue cleaved.

[0145] 4. G-CSF dimer pharmaceutical composition In some embodiments, a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., fbemalenograstim alfa) is formulated into a pharmaceutical composition hereafter referred to as the “G-CSF dimer pharmaceutical composition.” Any of the pharmaceutical compositions (and syringes containing them) described in PCT / CN2022 / 113560, filed on 19 August 2022, may be used in the “same-day administration” method described herein, the entirety of which is incorporated herein by reference.

[0146] In some embodiments, the G-CSF dimer pharmaceutical composition comprises (a) about 1 mg / mL to about 100 mg / mL of G-CSF molecules (e.g., G-CSF dimers described herein, e.g., G-CSF-Fc dimers, e.g., f-bemalenograstim alfa), (b) about 1 mM to about 50 mM of a buffering agent (e.g., sodium acetate), (c) about 0.1 mM to about 20 mM of a stabilizer (e.g., EDTA), (d) about 1% (w / v) to about 10% (w / v) of an isotonic agent (e.g., sorbitol or sucrose), and about 0.001% (w / v) to about 0.1% (w / v) of a surfactant (e.g., polysorbate 80 or polysorbate 20). In some embodiments, the pH of the G-CSF dimer pharmaceutical composition is about 4.2 to about 6.2, for example, about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2.

[0147] In some embodiments, the pharmaceutical composition contains a G-CSF dimer in a concentration of about 1 mg / mL to about 100 mg / mL, for example, a G-CSF dimer in a concentration of about 1 mg / mL to about 90 mg / mL, about 1 mg / mL to about 80 mg / mL, about 1 mg / mL to about 70 mg / mL, about 1 mg / mL to about 60 mg / mL, about 1 mg / mL to about 50 mg / mL, about 5 mg / mL to about 40 mg / mL, about 5 mg / mL to about 30 mg / mL, about 10 mg / mL to about 25 mg / mL, or about 20 mg / mL. In some embodiments, the pharmaceutical composition contains a G-CSF dimer in a concentration of about 18 mg / mL to about 22 mg / mL, for example, a G-CSF dimer in a concentration of about 20 mg / mL.

[0148] Examples of buffering agents, though not limited to these, include citrates, phosphates, acetates, succinates, tartrates, maleates, HEPES, tris, bicine, glycine, N-glycylglycine, carbonates, glycylglycine, lysine, arginine, histidine, and / or mixtures thereof. In some embodiments, the buffering agent is sodium acetate. In some embodiments, the pharmaceutical composition contains sodium acetate in amounts of about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, or about 10 mM.

[0149] In some embodiments, sodium acetate is a buffer that can be used to maintain the pH of the pharmaceutical composition at approximately pH 4.2 to approximately pH 6.2, for example, approximately pH 4.4 to approximately pH 6.0, approximately pH 4.6 to approximately pH 5.8, approximately pH 4.8 to approximately pH 5.8, approximately pH 4.8 to approximately pH 5.6, or approximately pH 5.0 to approximately pH 5.4. In some embodiments, the pH of the pharmaceutical composition is approximately pH 5.0 to approximately pH 5.4 (pH 5.2 ± 0.2).

[0150] Stabilizers can be added to improve the stability of pharmaceutical compositions and extend their shelf life. Examples of stabilizers, but not limited to, include glycine, alanine, glutamic acid, methionine, arginine, benzoic acid, citric acid, glycolic acid, lactic acid, malic acid, maleic acid, polyols (such as sorbitol, mannitol, and trehalose), surfactants, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylenediaminetriacetic acid (HEDTA), ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), metal ion stabilizers, and citrates. In some embodiments, the stabilizer is EDTA. In some embodiments, the pharmaceutical composition contains EDTA in any of the following concentrations: about 0.1 mM to about 20 mM, about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 2 mM, about 1 mM to about 2 mM, or about 1 mM. EDTA may function as a chelating agent, preservative, or stabilizer to prevent catalytic oxidation reactions in the formulation during processing and storage.

[0151] The pharmaceutical composition may contain one or more isotonic agents. The isotonic agents reduce local irritation caused by the pharmaceutical composition by preventing osmotic shock at the application site. Examples of isotonic agents, but not limited to, include sugar alcohols or polyols (such as mannitol or polyols), nonionic surfactants (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80), and sugars (e.g., sucrose). In some embodiments, the isotonic agent is sorbitol. In some embodiments, the pharmaceutical composition contains about 1% to about 10% (w / v), about 3% to about 10% (w / v), about 5% to about 10% (w / v), about 5% to about 8% (w / v), about 5% to about 7% (w / v), or about 5% to about 10% (w / v) of sorbitol. In some embodiments, the isotonic agent is sucrose. In some embodiments, the pharmaceutical composition contains about 5% to about 15% (w / v), about 7% to about 15% (w / v), about 8% to about 15% (w / v), about 9% to about 12% (w / v), about 9% to about 10% (w / v), or about 9% (w / v) sorbitol.

[0152] Nonionic surfactants such as polysorbates, including polysorbate (Tween®) 20 and polysorbate (Tween®) 80; polyoxamers, including poloxamer 184 and 188; pluronic® polyols; and other ethylene / polypropylene block polymers stabilize pharmaceutical compositions during processing and storage by reducing interfacial interactions and prevent protein adsorption. In some embodiments, the pharmaceutical composition contains polysorbate 20 (PS20). In some embodiments, the pharmaceutical composition contains approximately 0.001% (w / v) to approximately 0.1% (w / v), approximately 0.001% (w / v) to approximately 0.08% (w / v), approximately 0.001% (w / v) to approximately 0.05% (w / v), approximately 0.005% (w / v) to approximately 0.02% (w / v), approximately 0.005% (w / v) to approximately 0.01% (w / v), or approximately 0.01% (w / v) of polysorbate 20.

[0153] In some embodiments, the G-CSF dimer pharmaceutical composition comprises (a) about 20 mM G-CSF dimer (e.g., any G-CSF dimer described herein, e.g., a G-CSF-Fc dimer such as f-bemalenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.2 (e.g., pH 5.2 ± 0.2).

[0154] In some embodiments, the G-CSF dimer pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the G-CSF dimer pharmaceutical composition in the syringe is approximately 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0155] G-CSF dimer pharmaceutical compositions can be administered to an individual (e.g., a human, e.g., an individual with cancer) via various routes, including intravenous, intra-arterial, intraperitoneal, intravascular, intramuscular, subcutaneous, transmucosal, and transdermal. In some embodiments, a sustained-release formulation of the pharmaceutical composition can be used. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. When administered subcutaneously, such pharmaceutical compositions are typically administered in volumes of less than about 2.0 mL, or about 1.5 mL, or about 1 mL, or about 0.5 mL per injection site. In some embodiments, the injection volume is 1 mL. [Examples]

[0156] The following examples are intended purely to illustrate the present invention and should therefore not be considered to limit it in any way. The following examples and detailed description are provided for illustrative purposes only and not to limit it.

[0157] Example 1: Preparation of G-CSF-Fc dimer and formulation The exemplary G-CSF dimer is a homodimer of two G-CSF-Fc monomer subunits, each containing a human G-CSF monomer (SEQ ID NO: 1), a GS linker (SEQ ID NO: 12), and a human IgG2-derived Fc fragment (SEQ ID NO: 2) from the N-terminus to the C-terminus, with each monomer subunit containing the amino acid sequence of SEQ ID NO: 6. The two monomer subunits are linked by two pairs of disulfides. This exemplary G-CSF dimer is also referred to herein as G-CSF-Fc dimer F-627 or f-bemalenograstim alfa and is illustrated in Figure 1. The hIgG2-derived Fc fragment has the substitution P297S when numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomer subunit, which corresponds to the substitution P331S located in the CH2 domain according to EU numbering. Each G-CSF-Fc monomer subunit has one N-linked glycosylation site at N263 when the entire polypeptide chain of the G-CSF-Fc monomer subunit is numbered from the N-terminus, which corresponds to N297 located in the CH2 domain according to EU numbering. Each G-CSF-Fc monomer subunit also has one O-linked glycosylation site at T133 of the hG-CSF monomer. F-bemalenograstim alfa has a predicted molecular weight of approximately 89,497 Da (calculated based on the entire amino acid sequence, excluding carbohydrates).

[0158] F-bemalenograstim alpha, whose entire contents are incorporated herein by reference, was constructed and produced in Chinese hamster ovary cells by the method described in US8557546B2. The intact molecule of F-bemalenograstim alpha has a molecular weight of approximately 93.4 kD.

[0159] The liquid formulation of f-bemalenograstim alfa is manufactured as a solution packaged in sterile, single-use, preservative-free pre-filled syringes for convenient subcutaneous injection, delivering a target of 1 mL (20 mg of f-bemalenograstim alfa) per pre-filled syringe. Each pre-filled syringe contains 1 mL of liquid formulation containing 20 mg of f-bemalenograstim alfa, 10 mM sodium acetate, 1 mM EDTA, 5% sorbitol, and 0.01% polysorbate 20 (w / v), with a pH of 5.2 ± 0.2. The formulation and preparation method are described in PCT / CN2022 / 113560, filed on 19 August 2022, and the entire contents thereof are incorporated herein by reference.

[0160] Example 2: Efficacy study of G-CSF-Fc dimers with different drug regimens in rats with docetaxel / cyclophosphamide-induced neutropenia The efficacy of a representative G-CSF-Fc dimer (efbemarenograstim alfa) was compared with pegfilgrastim (Neulasta®) in different drug regimens in a chemotherapy-induced neutropenia rat model.

[0161] 1.Material Preparation of test solution To prepare a 60 μg / mL efbemalenograstim alfa solution for subcutaneous administration, 10 μL of efbemalenograstim alfa (20 mg / mL, in a pre-filled syringe) was vortexed with 3.323 mL of Vehicle 1 until a homogeneous suspension was achieved. Vehicle 1 contained 10 mM sodium acetate, 1 mM EDTA-Na2, 5% sorbitol, and 0.01% polysorbate 20 (w / v). To prepare a 20 μg / mL pegfilgrastim solution for subcutaneous administration, 10 μL of pegfilgrastim (10 mg / mL, Neulasta®, Amgen) was vortexed with 4.99 mL of Vehicle 2 until a homogeneous suspension was achieved. Vehicle 2 was the same formulation buffer as Neulasta®, containing 35 mg of acetate, 2 mg of polysorbate 20, 2 mg of sodium, and 3 g of sorbitol in 60 mL of sterile water.

[0162] Preparation of neutropenia-inducing agent solution To prepare a 0.8 mg / mL docetaxel solution, 80 mg of docetaxel was mixed with 10 mL of DMSO and 90 mL of 20% SBE-β-CD (in physiological saline) by vortexing and sonication until a homogeneous solution was achieved. To prepare a 6.4 mg / mL cyclophosphamide (CPA) solution, 640 mg of CPA was mixed with 100 mL of sterile water by sonication and vortexing until a homogeneous solution was achieved.

[0163] 2. Method Testing system [Table 3]

[0164] treatment The animals were randomly divided into 10 groups (G1-G10) based on body weight, with 5 animals in each group. To induce neutropenia, 4 mg / kg of docetaxel and 32 mg / kg of cyclophosphamide (collectively referred to as "TC") were administered intraperitoneally to rats in groups G2-G10 (day 0). Animals in group G2 were treated in Vehicle 1 0 hours after TC treatment (i.e., simultaneously with TC treatment). Animals in groups G3-G6 were treated with pegfilgrastim at a dose of 100 μg / kg (equivalent to 100 μg / kg in terms of G-CSF monomers), and animals in groups G7-G10 were treated with efbemarenograstim alfa at a dose of 300 μg / kg, equivalent to 124 μg / kg in terms of G-CSF monomers including O-linked glycosylation sites. Pegfilgrastim or fbemalenograstim alfa was administered subcutaneously once at 0 hours (G3, G7; i.e., simultaneously with TC treatment), 2 hours (G4, G8), 5 hours (G5, G9), or 24 hours (G6, G10) after TC treatment. Animals in G1 were controls that did not receive either TC or the test substance treatment. The dose was 5 mL / kg. The design of the test groups is shown in Table 2. [Table 4]

[0165] Observation and measurement Absolute Neutrophil Count (ANC) Profile: Whole blood samples were collected from test animals at -1 day before TC treatment (used as the ANC for day 0) and at 0.25, 1, 2, 3, 4, 5, 6, 7, and 8 days after TC treatment. Blood samples were dispensed into EDTA-K2 coated 1.5 mL centrifuge tubes for complete blood cell count (CBC) analysis. Standard hematological tests were performed at room temperature using ADVIA® 2120i (Siemens).

[0166] Neutropenia duration (DN) profile: The primary endpoint of this study was determined from the DN, which was based on the neutrophil level cutoff value (overall mean neutrophil level) calculated from animals in the control group (G1).

[0167] 3.Results ANC Profile The time-course of the number of neutrophils is shown in Fig. 2. In the vehicle treatment group (G2; “TC + vehicle 1”), the number of neutrophils significantly decreased at 24 hours after TC treatment compared with the control group (G1; “normal”), indicating that a neutropenia model induced by TC was successfully established. Treatment with pegfilgrastim or efimerenograstim alpha showed that the number of neutrophils recovered to or exceeded the normal ANC level. At Nadia (day 3, i.e., 72 hours after TC treatment), when administered on the same day as chemotherapy (0 hours, 2 hours, and 5 hours after TC treatment), the mean ANC values of rats administered efimerenograstim alpha were approximately 3.5-fold, 1.4-fold, and 2.1-fold those of rats administered pegfilgrastim, respectively. At Nadia on day 3, when the test article was administered 24 hours after TC treatment, the mean ANC levels of rats administered efimerenograstim alpha and pegfilgrastim were similar (1.10×10 9 / L and 1.21×10 9 / L, respectively). Furthermore, from 96 hours to 192 hours after chemotherapy, all rats with neutropenia administered efimerenograstim alpha (G7 - G10) had significantly higher ANC levels than rats with neutropenia administered pegfilgrastim. When efimerenograstim alpha was administered 24 hours after TC treatment, two Nadia (day 1 and day 3) were observed in ANC, whereas when efimerenograstim alpha was administered on the same day as chemotherapy (e.g., 0 hours, 2 hours, or 5 hours after TC treatment), a smoother ANC profile was shown (Nadia only once on day 3).

[0168] DN Profile In this study, rats were diagnosed with neutropenia if their neutrophil levels were lower than the overall mean of neutrophil levels in the control group (G1). Docetaxel and cyclophosphamide (TC) administration resulted in neutropenia lasting approximately 7.4 days. As shown in Table 3, the DN values ​​for efbemarenograstim alfa or pegfilgrastim administered 24 hours after chemotherapy were 2.0 days and 1.8 days, respectively. As the interval between chemotherapy and the administration of the test drug shortened (5 hours or 2 hours after TC treatment, or simultaneously with TC treatment (0 hours)), the DN in the pegfilgrastim group increased to approximately 1.6–2.8 days. In contrast, the DN in the efbemarenograstim alfa group decreased to approximately 0.4–0.8 days.

[0169] Therefore, when G-CSF-Fc dimers are administered on the same day as chemotherapy (e.g., TC) (e.g., 0 hours (simultaneous), 2 hours, or 5 hours after TC treatment), they can treat and / or prevent chemotherapy-induced neutropenia with far greater efficacy compared to pegfilgrastim administered with an equivalent amount of G-CSF monomer. This demonstrates that G-CSF-Fc dimers may offer a flexible administration schedule less than 24 hours after chemotherapy. Furthermore, administering G-CSF-Fc dimers less than 24 hours after chemotherapy (e.g., 0 hours (simultaneous), or 2 or 5 hours after chemotherapy) is thought to be more effective in treating and / or preventing neutropenia compared to a 24-hour administration regimen. [Table 5]

[0170] Example 3: A study to evaluate the duration of severe neutropenia after same-day administration of fbemarenograstim alfa in breast cancer patients undergoing myelosuppressive chemotherapy. The primary objective of this study is to evaluate the duration of severe neutropenia following same-day administration of efbemarenograstim alfa 20 mg in breast cancer patients undergoing myelosuppressive chemotherapy.

[0171] 1. Test evaluation items Primary efficacy endpoints The primary endpoint was grade 4 (severe) neutropenia (ANC < 0.5 × 10⁻¹⁰). 9 The duration of the ANC is / L), and the target group is in cycle 1 of chemotherapy treatment, where ANC < 0.5 × 10 9 It is defined as the number of days indicated by / L.

[0172] Secondary efficacy endpoints The effectiveness evaluation items are as follows: • Incidence of Grade 4 neutropenia in Cycle 1. • Incidence and duration of Grade 4 neutropenia in Cycle 1. • Grade 3 or 4 neutropenia in Cycle 1 (ANC < 1.0 × 10, respectively) 9 / L and ANC < 0.5 × 10 9 The incidence and duration of / L). • Incidence of febrile neutropenia (FN) in Cycle 1. Febrile neutropenia is defined as a single oral temperature reading of 38.3°C (101°F) or higher, or a temperature exceeding 38.0°C (100.4°F) for more than one hour, and a 0.5 × 10⁻¹⁰ day rate. 9 This is defined as when an ANC less than / L is shown. • Incidence of hospitalization due to febrile neutropenia or any infection during Cycle 1. • The depth of ANC Nadia in Cycle 1. • Number of days to ANC recovery after chemotherapy in Cycle 1. Recovery is defined as the number of days after the expected ANC nadia, when the ANC reaches 2.0 × 10⁶. 9 Defined as / L or higher.

[0173] Safety evaluation items The following safety evaluation items will be monitored: Adverse events (AEs). Vital signs. • Clinical test values. • Physical examination. • Concomitant medications (especially painkillers for bone pain).

[0174] 2. Test Design This study is a randomized, schedule-exploratory, three-group, open-label, multicenter Phase I trial to evaluate same-day administration of 20 mg of efbemarenograstim alfa (subcutaneous) after IV infusion of chemotherapy. In this trial, myelosuppressive TC chemotherapy (docetaxel 75 mg / m²) is used as neoadjuvant or adjuvant therapy. 2 +Cyclophosphamide 600mg / m² 2 Approximately 45 women (15 in each group) with stage I-III invasive breast cancer who are scheduled to receive chemotherapy will be enrolled. This study is for patients scheduled to receive at least four cycles of 21-day chemotherapy. While some patients may be scheduled for more than four cycles, participation in this study will be limited to the first four cycles.

[0175] treatment On day 1 of cycle 1, patients are randomized in a 1:1:1 ratio to receive efbemalenograstim alfa 0.5 hours, 3 hours, and 5 hours after TC treatment. In cycles 2-4, all treatment groups receive efbemalenograstim alfa 24 hours after TC administration on day 1 of each cycle.

[0176] group 1 Administer a fixed-dose pre-filled syringe of fbemalenograstim alfa 20 mg 0.5 hours after TC administration on day 1 of cycle 1. In cycles 2-4, administer fbemalenograstim alfa 24 hours after TC administration on day 1 of each cycle.

[0177] group 2 Administer a fixed-dose pre-filled syringe of fbemalenograstim alfa 20 mg three hours after TC treatment on day 1 of cycle 1. In cycles 2-4, administer fbemalenograstim alfa 24 hours after TC administration on day 1 of each cycle.

[0178] group 3Administer a 20 mg fixed-dose pre-filled syringe of fbemalenograstim alfa 5 hours after TC treatment on day 1 of cycle 1. In cycles 2-4, administer fbemalenograstim alfa 24 hours after TC administration on day 1 of each cycle.

[0179] Effectiveness evaluation All ANC measurements used for analysis are performed in the central laboratory. However, if a sample from the central laboratory is missing or of poor quality during the first chemotherapy cycle, ANC values ​​from a local laboratory will be used. In addition, local laboratory test results may be used to monitor the safety of subjects during the study period, and these results will be reported if associated with adverse events (AEs).

[0180] Safety evaluation Temperature will be measured on days 1, 2, and 3 of each cycle. If body temperature exceeds 38.0°C at any point, the subject must measure their temperature again one hour later. If the temperature remains above 38.0°C for more than one hour, the subject must contact the person in charge at the clinical trial site.

[0181] Adverse events (AEs) will be monitored and evaluated throughout the study period in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v5.0). Safety monitoring will include vital signs, physical examination, hematological tests, serological chemistry, and urinalysis.

[0182] Pharmacokinetic (PK) assessment For the subjects, blood samples will be collected before administration on days 2, 4, and 8 of chemotherapy cycles 1 and 3 to evaluate the drug concentration in the serum.

[0183] Blood samples for hematological testing will be taken daily for the first 10 days of Cycle 1, and then on the first day of each cycle from Cycle 2 to Cycle 4.

[0184] End of Treatment (EOT) Participants will visit the end-of-treatment (EOT) clinic approximately 20 days after the final administration of the study drug, and will then have a follow-up visit approximately 6 months after the date of their final administration of the study drug.

[0185] 3. Inclusion Criteria The inclusion criteria are as follows: • Presentation of an informed consent document signed and dated by the patient, indicating that the patient was informed about all important aspects of the trial. • Female, 18 years of age or older. She has been diagnosed with stage I-III breast cancer. The target group is those receiving TC chemotherapy (docetaxel 75 mg / m²) as neoadjuvant or postadjuvant therapy. 2 +Cyclophosphamide 600mg / m² 2 I am scheduled to take four cycles of this. • The East Coast Cancer Group (ECOG) performance status is 2 or lower. • ANC is 2.0x10 9 Hemoglobin levels above 11.0 g / dL and platelet counts above 100 × 10 9 / L or higher. • Indicates adequate renal, hepatic, and cardiac function [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase levels are less than 2.5 times the upper limit of normal (ULN)]. Total bilirubin and serum creatinine levels are less than 1.5 times the ULN. All participants must agree to use at least one method of contraception from the following: intrauterine devices, implantable progesterone preparations, intramuscular progesterone injections, or oral contraceptives. These must be initiated at least one month prior to the first visit and continued throughout the study period. Contraceptive patches or condoms with spermicides are also acceptable methods of contraception, provided they are used continuously throughout the study period.

[0186] 4. Treatment period The trial treatment period totals approximately 12 weeks, including a screening phase (3 weeks), with the end-of-treatment (EOT) visit occurring approximately 20 days after the final trial dose.

[0187] Participants will receive treatment according to randomization to a treatment group on day 1 of the initial chemotherapy cycle. Participants will receive treatment on day 1 of each subsequent chemotherapy cycle, for a maximum of four cycles of 21 days each. To initiate full-dose chemotherapy on day 1 of the next cycle (day 22 of the previous cycle), patients must have a baseline hemoglobin of at least 11.0 g / dL and an ANC of 2.0 × 10⁻⁶. 9 The platelet count exceeds / L and is 80 × 10 9 A value exceeding / L is recommended.

[0188] Clinical evaluation will be conducted for all subjects during the screening period (-21 days to -1 day).

[0189] Chemotherapy cycle 1 Depending on the treatment group, participants will receive efemarenograstim alfa 0.5 hours, 3 hours, or 5 hours after chemotherapy administration.

[0190] During the first chemotherapy cycle, and after chemotherapy, subjects will visit the clinical trial site daily for blood sampling to track the progression of ANC. Subjects will visit the site from day 2 to day 10, and thereafter, if the ANC level is 2.0 × 10 after NADI, 9Patients will visit the hospital daily until their ANC level reaches 1 / L or higher, and then three days later, they will visit again for the final ANC measurement of their chemotherapy cycle.

[0191] Chemotherapy cycles 2, 3, and 4 In each cycle from cycle 2 to 4, participants will visit the hospital 24 hours after chemotherapy administration for administration of the study drug. The actual visit date may vary slightly from cycle to cycle, as it depends on the participant's chemotherapy schedule.

[0192] Example 4: A study to evaluate the efficacy and safety of "same-day administration" and "next-day administration" of fbemarenograstim alfa in breast cancer patients undergoing myelosuppressive chemotherapy. The primary objective of this study is to compare the effect of "same-day administration" (less than 24 hours after chemotherapy) and "next-day administration" (24 hours or more after chemotherapy) of 20 mg of efbemarenograstim alfa on the duration of severe neutropenia in breast cancer patients undergoing myelosuppressive chemotherapy.

[0193] 1. Test evaluation items Primary efficacy endpoints The primary endpoint was grade 4 (severe) neutropenia (ANC < 0.5 × 10⁻¹⁰). 9 The duration of the ANC is / L), and the target group is in cycle 1 of chemotherapy treatment, where ANC < 0.5 × 10 9 It is defined as the number of days indicated by / L.

[0194] Secondary efficacy endpoints The effectiveness evaluation items are as follows: • Duration of Grade 4 neutropenia in cycles 2-4. • Incidence of Grade 4 neutropenia in cycles 1-4. • Grade 3 or 4 neutropenia in cycles 1-4 (ANC < 1.0 × 10, respectively) 9 / L and ANC < 0.5 × 10 9 Duration of the effect ( / L). · Incidence of grade 3 or 4 neutropenia in cycles 1 - 4 (ANC < 1.0×10 9 / L and ANC < 0.5×10 9 / L, respectively). · Incidence of grade 4 febrile neutropenia in cycles 1 - 4. Febrile neutropenia is defined as a single oral temperature of 38.3°C (101°F) or higher, or a temperature exceeding 38.0°C (100.4°F) persisting for more than 1 hour on the same day, and an ANC of less than 0.5×10 9 / L on the same day. · Frequency of hospitalization due to febrile neutropenia or any infectious disease in cycles 1 - 4. · Depth of ANC nadir in cycles 1 - 4. · Number of days until ANC recovery after chemotherapy in cycles 1 - 4. Recovery is defined as when the ANC reaches 2.0×10 9 / L or more after the expected ANC nadir.

[0195] Safety evaluation items , the following safety evaluation items are monitored: · Adverse events. · Vital signs. · Clinical laboratory values. · Physical examinations. · Concomitant medications (especially analgesics for bone pain).

[0196] · Randomized, open - label, multi - center, parallel - group, phase III trial to evaluate the efficacy and safety of "same - day administration" and "next - day administration" of 20 mg of pegfilgrastim alpha administered subcutaneously to breast cancer patients receiving myelosuppressive chemotherapy. In this trial, myelosuppressive TC chemotherapy (docetaxel 75 mg / m<000090... 2 + cyclophosphamide 600 mg / m 2Approximately 140 female subjects (70 in each group) with stage I-III invasive breast cancer who are scheduled to receive

[0197] treatment are enrolled. The subjects of this trial are those who are scheduled to receive at least 4 cycles of chemotherapy with a 21-day cycle. The subjects may be scheduled to receive chemotherapy beyond 4 cycles, but participation in this trial is limited to the first 4 cycles of the subjects.

[0198] group 1 : Pegfilgrastim alfa, 20 mg fixed-dose prefilled syringe, is administered at X hours (X is selected from 0.5 hour, 3 hours or 5 hours after TC treatment based on the results of the phase I trial described in Example 3) after TC treatment on day 1 of each cycle (「same-day administration」), or is administered 24 hours after TC treatment on day 2 of each cycle (「next-day administration」).

[0199] group 2 : Pegfilgrastim alfa, 20 mg fixed-dose prefilled syringe, is administered 24 hours after TC treatment on day 2 of each cycle.

[0200] Effectiveness evaluation All ANC measurements used for analysis are performed at the central laboratory. However, if the subject's sample at the central laboratory is missing or has quality problems during the first chemotherapy cycle, the ANC value at the local laboratory is used. Furthermore, for the purpose of monitoring the safety of the subjects during the trial, the test results at the local laboratory may be used, and these results are reported if they are related to AE.

[0201] Safety evaluation Temperature will be measured on days 1, 2, and 3 of each cycle. If body temperature exceeds 38.0°C at any point, the subject must measure their temperature again one hour later. If the temperature remains above 38.0°C for more than one hour, the subject must contact the person in charge at the clinical trial site.

[0202] Adverse events (AEs) will be monitored and evaluated throughout the study period in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v5.0). Safety monitoring will include vital signs, physical examination, hematological tests, serological chemistry, and urinalysis.

[0203] PK rating For the subjects, blood samples will be collected before administration on days 2, 4, and 8 of chemotherapy cycles 1 and 3 to evaluate the drug concentration in the serum.

[0204] End of Treatment (EOT) Participants will remain in the randomized trial group throughout the subsequent three chemotherapy cycles. Participants will visit the end-of-treatment (EOT) approximately 20 days after the final administration of the investigational drug.

[0205] 3. Inclusion Criteria The inclusion criteria are as follows: • Presentation of an informed consent document signed and dated by the patient, indicating that the patient was informed about all important aspects of the trial. • Female, 18 years of age or older. She has been diagnosed with stage I-III breast cancer. The target group is those receiving TC chemotherapy (docetaxel 75 mg / m²) as neoadjuvant or postadjuvant therapy. 2 +Cyclophosphamide 600mg / m² 2 I am scheduled to take four cycles of this. • ECOG performance status is 2 or lower. • ANC is 2.0x10 9 Hemoglobin levels above 11.0 g / dL and platelet counts above 100 × 10 9 / L or higher. • Indicates adequate renal, hepatic, and cardiac function [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase levels are less than 2.5 times the upper limit of normal (ULN)]. Total bilirubin and serum creatinine levels are less than 1.5 times the ULN. All participants must agree to use at least one method of contraception from the following: intrauterine devices, implantable progesterone preparations, intramuscular progesterone injections, or oral contraceptives. These must be initiated at least one month prior to the first visit and continued throughout the study period. Contraceptive patches or condoms with spermicides are also acceptable methods of contraception, provided they are used continuously throughout the study period.

[0206] 4. Treatment period The trial treatment period totals approximately 12 weeks, including a screening phase (3 weeks), with the end-of-treatment (EOT) visit occurring approximately 20 days after the final trial dose.

[0207] Participants will receive treatment according to randomization to a treatment group on day 1 of the initial chemotherapy cycle. Participants will receive treatment on day 1 of each subsequent chemotherapy cycle, for a maximum of four cycles of 21 days each. To initiate full-dose chemotherapy on day 1 of the next cycle (day 22 of the previous cycle), patients must have a baseline hemoglobin of at least 11.0 g / dL and an ANC of 2.0 × 10⁻⁶. 9 If the platelet count exceeds / L and is 100 × 10⁶ 9 A value exceeding / L is recommended.

[0208] Clinical evaluation will be conducted for all subjects during the screening period (-21 days to -1 day).

[0209] Chemotherapy cycle 1 Subjects receive 20 mg of pegfilgrastim alpha either X hours after the TC treatment on Day 1 (the "same-day administration" group) or 24 hours after the TC treatment on Day 2 (the "next-day administration" group), depending on the treatment group.

[0210] During the first chemotherapy cycle, subjects come to the trial facility for daily blood sampling to track the change in ANC after chemotherapy. Subjects come to the hospital on Days 2 to 10, and then, until the ANC level reaches 2.0×10 9 / L or more after nadir, they come to the hospital daily, and then subjects come to the hospital 3 days later for the final ANC measurement of the chemotherapy cycle.

[0211] Chemotherapy cycles 2, 3, and 4 Subjects remain in the randomized study group and receive 20 mg of pegfilgrastim alpha after chemotherapy. As described above, the actual hospital visit days may vary slightly for each cycle because they depend on the individual chemotherapy schedule of the subject.

[0212] In each of Cycles 2, 3, and 4, subjects come to the trial facility for blood sampling every other day to track the change in ANC after chemotherapy. Subjects come to the hospital every other day from Day 3 to Day 11, and then, until the ANC level reaches 2.0×10 9 / L or more after nadir, they come to the hospital daily, and then subjects come to the hospital 3 days later for the final ANC measurement of the chemotherapy cycle.

[0213] If the subject's ANC level is less than 0.5×10 9 / L in two consecutive hospital visits, the subject comes to the hospital the next day and then must come to the hospital daily for ANC blood sampling until the ANC level reaches 2.0×10 9 / L or more.

[0214] Example 5: A Phase III, randomized, multi-center, open-label, fixed-dose, Neulasta (registered trademark) active-controlled clinical trial of pegfilgrastim alpha in female breast cancer patients receiving myelosuppressive chemotherapy This example demonstrates that 20 mg of an exemplary G-CSF dimer (f-bemalenograstim alfa), administered 24 hours after chemotherapy, is effective in treating chemotherapy-induced neutropenia in humans, and that its effect is equivalent to that of the long-acting G-CSF drug pegfilgrastim (Neulasta®).

[0215] A randomized, open-label, active-controlled Phase III trial was conducted to compare the efficacy and safety of ephemarenograstim alfa with that of pegfilgrastim (Neulasta®) in breast cancer patients undergoing chemotherapy. The primary objective of this trial was to evaluate the safety and efficacy of ephemarenograstim alfa administered as a single fixed-dose (20 mg) pre-filled syringe (PFS) compared with Neulasta® (6 mg) in the first chemotherapy cycle. A total of 393 breast cancer patients were enrolled in this trial, of which 197 received ephemarenograstim alfa 20 mg and 196 received pegfilgrastim 6 mg. Both groups received docetaxel 75 mg / m² on day 1 of each cycle. 2 and cyclophosphamide 600 mg / m² 2 The patient received four cycles of chemotherapy (21 days per cycle). Efbemarenograstim alfa and the control drug pegfilgrastim were administered once subcutaneously on day 2 of each chemotherapy cycle (i.e., 24 hours after the administration of chemotherapy).

[0216] The trial results met the pre-specified primary efficacy endpoint. As shown in Table 4, the primary efficacy endpoint (mean duration of grade 4 neutropenia in cycle 1) was 0.2 days in both groups (mean difference, 0.0 days; 95% CI, -0.1 to 0.1). The difference in the incidence of febrile neutropenia (FN, p=0.62) and the number of days of intravenous antibiotic use was not statistically significant between the two groups over all four chemotherapy cycles (p=0.05). One patient in each group was hospitalized with FN or either infection. The overall incidence of infection was 7.1% in the efbemarenograstim alfa group and 8.7% in the pegfilgrastim group (p=0.58). Secondary efficacy endpoint analyses also showed that efbemarenograstim alfa was comparable to pegfilgrastim. The incidence of grade 4 neutropenia in cycle 4 was 3 / 186 (1.6%) in the efbemarenograstim alfa group and 10 / 188 (5.3%) in the pegfilgrastim group, respectively. The incidence of grade 4 neutropenia in cycle 4 was lower in the efbemarenograstim alfa group than in the pegfilgrastim group (p=0.05), indicating that efbemarenograstim alfa has a favorable therapeutic effect in the treatment of chemotherapy-induced neutropenia. [Table 6]

[0217] Example 6: Efficacy study of G-CSF-Fc dimers with different drug regimens in rats with docetaxel / cyclophosphamide-induced neutropenia The efficacy of an exemplary G-CSF-Fc dimer (efbemarenograstim alfa) was compared to efrapegrastim in different drug regimens in a chemotherapy-induced neutropenia rat model.

[0218] 6.1.Materials Preparation of test solution To prepare a 66.6 μg / mL f-bemalenograstim alfa solution for subcutaneous administration, 33.3 μL of f-bemalenograstim alfa (20 mg / mL, in a pre-filled syringe) was vortexed with 9.97 mL of Vehicle 1 until a homogeneous suspension was achieved. To prepare a 30 μg / mL f-bemalenograstim alfa solution for subcutaneous administration, 2 mL of f-bemalenograstim alfa solution (66.6 μg / mL) was vortexed with 2.44 mL of Vehicle 1 until a homogeneous suspension was achieved. Vehicle 1 contained 10 mM sodium acetate, 1 mM EDTA-Na2, 5% sorbitol, and 0.01% polysorbate 20 (w / v).

[0219] To prepare a 44 μg / mL efrapegrastim solution for subcutaneous administration, 10 μL of efrapegrastim (22 mg / mL) was vortexed with 4.99 mL of Vehicle 2 until a homogeneous suspension was achieved. Vehicle 2 is the same formulation buffer containing 25.2 mg of citric acid monohydrate, 300 mg of mannitol, 7.2 mg of polysorbate 80, and 52.6 mg of sodium chloride in 6 mL of sterile water.

[0220] Preparation of neutropenia-inducing agent solution To prepare a 0.8 mg / mL docetaxel solution, 80 mg of docetaxel was mixed with 10 mL of DMSO and 90 mL of 20% SBE-β-CD (in physiological saline) by vortexing and sonication until a homogeneous solution was achieved. To prepare a 6.4 mg / mL cyclophosphamide (CPA) solution, 640 mg of CPA was mixed with 100 mL of sterile water by sonication and vortexing until a homogeneous solution was achieved.

[0221] 6.2. Method Testing system [Table 7]

[0222] treatment The animals were randomly divided into 11 groups (G1-G11) based on body weight, with 8 animals in each group. On day 0, 4 mg / kg of docetaxel and 32 mg / kg of cyclophosphamide (collectively referred to as "TC") were administered intraperitoneally to rats in groups G2-G11 to induce neutropenia. Animals in group G2 were administered vehicle 1 0 hours after TC treatment (i.e., simultaneously with TC treatment). Animals in groups G3-G5 were treated with efrapegrastim at a dose of 220 μg / kg (equivalent to 62 μg / kg in terms of G-CSF monomers), and animals in groups G6-G8 and G9-G11 were treated with efbemarenograstim alfa at doses of 150 and 333 μg / kg, respectively (equivalent to 62 and 138 μg / kg in terms of G-CSF monomers including O-linked glycosylation sites). The clinical dose of efrapegrastim is 220 μg / kg (assuming a human body weight of 60 kg, this is 13.2 mg per single dose, 13.2 mg / 60 kg = 220 μg / kg). The clinical dose of efbemarenograstim alfa is 333 μg / kg (assuming a human body weight of 60 kg, this is 20 mg per single dose, 20 mg / 60 kg = 333 μg / kg). Efrapegrastim or efbemarenograstim alfa was administered subcutaneously as a single dose at 0 hours (G3, G6, G9; i.e., simultaneously with TC treatment), 2 hours (G4, G7, G10), or 5 hours (G6, G8, G11) after TC treatment. Animals in G1 were controls that did not receive either TC or the test substance treatment. The dose was 5 mL / kg. The design of the test groups is shown in Table 6. [Table 8]

[0223] Observation and measurement Absolute Neutrophil Count (ANC) Profile: Whole blood samples were collected from test animals at -1 day before TC treatment (used as the ANC for day 0) and at 0.25, 1, 2, 3, 4, 5, 6, 7, and 8 days after TC treatment. Blood samples were dispensed into EDTA-K2 coated 1.5 mL centrifuge tubes for complete blood cell count (CBC) analysis.

[0224] Neutropenia duration (DN) profile: The primary endpoint of this study was determined from the DN, which was based on the neutrophil level cutoff value (overall mean neutrophil level) calculated from animals in the control group (G1).

[0225] In at least one embodiment, animals subcutaneously administered ephebemarenograstim alfa at 150 μg / kg or 333 μg / kg (corresponding to 62 μg / kg and 138 μg / kg of G-CSF monomers, respectively) at 0, 2, or 5 hours after TC administration showed a comparable or shorter duration of neutropenia compared to animals administered 220 μg / kg of ephrapegrastim (corresponding to 62 μg / kg of G-CSF monomers). The findings of this study support further investigation of same-day administration of ephebemarenograstim alfa after chemotherapy in patients.

[0226] Example 7: A clinical trial evaluating the efficacy and safety of same-day subcutaneous administration of efbemarenograstim alfa after R-CHOP-like chemotherapy in patients with non-Hodgkin lymphoma. A clinical trial will be conducted to evaluate the feasibility of same-day administration of efebemarenograstim alfa in patients receiving R-CHOP-like chemotherapy (R: rituximab, C: cyclophosphamide (e.g., CYTOXAN®), H: doxorubicin (e.g., ADRIAMYCIN®), O: vincristine (e.g., ONCOVIN®), P: prednisone). The primary objective of this trial is to evaluate the efficacy of same-day administration of efebemarenograstim alfa 20 mg in non-Hodgkin lymphoma patients receiving R-CHOP-like chemotherapy. 7.1. Drug Information [Table 9]

[0227] 7.2. Test Evaluation Items Primary efficacy endpoints The primary endpoint is the incidence of grade 4 (severe) neutropenia (absolute neutrophil count (ANC) < 0.5 × 10⁹ / L) during cycle 1 of chemotherapy.

[0228] Secondary efficacy endpoints The secondary outcome measures are as follows: • Grade 3 or 4 neutropenia in all cycles (ANC < 1.0 × 10, respectively) 9 / L and ANC < 0.5 × 10 9 The incidence rate of / L). • Grade 4 neutropenia in all cycles (ANC < 0.5 × 10 9 The incidence rate of ). • Duration of severe neutropenia (DSN) in Cycle 1. • Timing and depth of ANC nadia, and number of days to ANC recovery after chemotherapy in cycle 1. Recovery is when ANC reaches 1.0 × 10⁶ after ANC nadia. 9 Defined as / L or higher. • Incidence of febrile neutropenia (FN) in all cycles. Febrile neutropenia is defined as a single oral body temperature of 38.3°C (101°F) or higher, or a body temperature of 38.0°C (100.4°F) or higher for more than one hour, and an ANC of 0.5 × 10⁶ on the same day. 9 If it is less than / L, or if ANC is 1.0 × 10 9 Less than / L, and within the following 48 hours, 0.5 × 10 9 This is defined as the level dropping to / L. • Incidence of infection in adverse events (AEs) across all cycles. • Incidence and duration of intravenous antibiotics used in all cycles. • Incidence and duration of hospitalization due to febrile neutropenia or any infection in all cycles.

[0229] Safety evaluation items • Incidence of adverse events: Includes adverse events, serious adverse events, adverse events leading to death or withdrawal, adverse events classified by severity, adverse events related to the investigational drug, and serious unexpected adverse events; • Clinical laboratory values ​​including hematological tests, blood biochemistry, coagulation function, and urinalysis; • 12-lead electrocardiogram examination; Vital signs; • Physical examinations and other safety-related inspections; • Frequency of concomitant medication use 7.3. Test Plan

[0230] This study is planned to enroll approximately 40 patients. All patients will undergo 4 to 6 treatment cycles (every 21 days per cycle) for research, treatment, and evaluation. The study will be conducted in two phases. In the first phase, the first 4 patients will be enrolled and their tolerability in the first treatment cycle will be evaluated. If tolerability is acceptable, the study will proceed to the second phase, enrolling the remaining 36 patients. If tolerability is not acceptable, the principal investigator will need to make a comprehensive decision on whether to proceed with the subsequent study plan. The criterion for determining unacceptable tolerability is the occurrence of two or more grade 4 or higher adverse events (AEs) among the 4 patients that are judged to be related to efbemarenograstim alfa.

[0231] treatment Approximately 40 eligible NHL patients will receive subcutaneous administration of efbemarenograstim alfa 4–6 hours after chemotherapy. The treatment plan for each administration cycle is as follows: Day 1: Rituximab, 375 mg / m² 2 Intravenous infusion.

[0232] Day 2: Cyclophosphamide, 750 mg / m² 2 Intravenous infusion; doxorubicin, 50 mg / m² 2 Intravenous infusion; vincristine, 1.4 mg / m² 2(Maximum 2.0 mg), intravenous infusion. Efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, subcutaneously 4-6 hours after chemotherapy. Days 2-6: Prednisone or prednisolone 100 mg is administered orally.

[0233] If some of the chemotherapeutic agents in the R-CHOP regimen described above are unavailable at certain research centers for specific reasons, the principal investigator may make a comprehensive decision to replace these drugs with similar drugs, for example, vincristine with vincristine.

[0234] Criteria for discontinuing fbemarenograstim alfa administration: In subsequent chemotherapy cycles, the pre-chemotherapy neutrophil count was 30 × 10¹⁰ in association with the prophylactic use of efbemarenograstim alfa. 9 If the dose exceeds / L, discontinue administration of efbemarenograstim alfa; If any of the following events occur after administration of efbemarenograstim alfa, discontinue administration for the subsequent treatment cycle: ·Aortitis; • Glomerulonephritis; ·Acute respiratory distress syndrome; • Severe allergic reaction.

[0235] Effectiveness evaluation All ANC measurements used for analysis are performed in the central laboratory. Cycle 1: Blood samples are taken for hematological testing on days 1, 6, 7, 8, 9, 10, and 14 to monitor neutrophil count. On day 10, the ANC is still 1.0 × 10⁶. 9 If it is below / L, ANC is 1.0×10 9 Complete blood counts (CBCs) are required daily until the neutrophil count recovers to above / L. Cycles 2-6: Blood samples for hematological testing are taken on days 1, 8, 10, and 14 to monitor neutrophil counts.

[0236] Safety evaluation Body temperature will be measured daily. If body temperature exceeds 38.0°C at any point, it will be measured again after one hour. If the temperature remains above 38.0°C for more than one hour, the participant must contact the clinical trial site staff. If body temperature exceeds 38.3°C, the patient must immediately contact the clinical trial site staff.

[0237] Adverse events (AEs) will be monitored and evaluated throughout the study period in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v5.0).

[0238] Safety monitoring includes vital signs, physical examination, hematological tests, blood biochemistry, coagulation function, urinalysis, and electrocardiogram.

[0239] Pharmacokinetic (PK) assessment In Cycle 1, blood samples will be collected to evaluate serum and ebbemalenograstim alfa concentrations at various time points before R-CHOP-like chemotherapy and after ebbemalenograstim alfa administration.

[0240] End of Treatment (EOT) Patients will visit the hospital for end-of-treatment (EOT) approximately 21 (+7) days after the final dose.

[0241] 7.4. Patient Inclusion Criteria The patient inclusion criteria are as follows: • Presentation of an informed consent document signed and dated by the patient, indicating that the patient was informed about all important aspects of the trial. • Female, 18 years of age or older. • Diagnosed with NHL by histopathology and deemed appropriate by the principal investigator for at least four cycles of R-CHOP-like treatment (including initial treatment and relapse / refractory cases); • The East Coast Cancer Group (ECOG) performance status is 2 or lower. • Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are less than 2.5 times the upper limit of normal (ULN), indicating adequate renal, hepatic, and cardiac function; total bilirubin and serum creatinine are 1.5 times the ULN, or creatinine clearance is 60 mL / min or higher. • ANC is 1.5 x 10 9 Hemoglobin levels above 9.0 g / dL and platelet count of 75 × 10 9 / L or higher. • Infertile women, i.e., those who have been postmenopausal for at least one year or who have undergone sterilization (bilateral tubal ligation, bilateral oophorectomy, or hysterectomy); infertile patients must agree to use appropriate contraception from one month before the trial until 30 days after the trial ends.

[0242] Exclusion criteria • Patients who are currently using or are scheduled to use other antitumor drugs other than R-CHOP-like drugs; • Associated with other malignancies besides NHL; • Patients with relapsed / refractory condition who have previously received autologous or allogeneic hematopoietic stem cell transplantation; Patients with a history of acute myeloid leukemia, myelodysplastic syndrome, and sickle cell anemia; Patients with a history of splenic rupture; • Patients with infections requiring intravenous anti-infective therapy; • Patients with HIV infection; Patients with poorly controlled HBV infection (HBsAg positive and / or HBV DNA titer positive, HBc Ab positive) or HCV infection (HCV Ab positive); • Patients with immune disorders requiring immunosuppressive therapy; Patients with a history of severe allergy to human G-CSF or excipients in efbemarenograstim alfa prefilled syringe; • Pregnant or breastfeeding women; • Patients with alcohol or drug abuse that could affect compliance with the study; • Any other disease or condition that, at the discretion of the principal investigator, could prevent the patient from participating in this study, or that the investigational drug could harm the patient's health, or that could affect the determination of adverse events.

[0243] Discontinuation / suspension criteria Patients must discontinue the study drug treatment if any of the following conditions apply, and the discontinuation visit will be completed according to the study protocol. For patients who discontinue treatment early, blood for complete blood count (CBC) as specified in the cycle protocol will be collected to monitor changes in neutrophil count: • Occurrence of adverse events (multiple events possible) leading to discontinuation of efbemarenograstim alfa treatment, as determined by the principal investigator; • Risks / benefits of patients whom the principal investigator has comprehensively considered and deemed unsuitable for continued study; • R-CHOP-like regimens are no longer used for the treatment of lymphoma; ·pregnancy; • Patient's voluntary discontinuation; Loss of follow-up; ·death.

[0244] The reasons for termination and suspension must be recorded in the electronic case report form (CRF).

[0245] 7.5. Treatment period The trial treatment period is a maximum of approximately 24 weeks in total, including a screening period (3 weeks), a treatment period (12-18 weeks), and an EOT visit approximately 3 (+1) weeks after the final administration of the study drug.

[0246] A clinical evaluation will be performed for all patients during the screening period (-21 days to -1 day).

[0247] The following steps were taken in each cycle: Day 1 • A 12-lead electrocardiogram does not need to be remeasured on day 1 of cycle 1 if it was measured within 7 days prior to day 1 of cycle 1. For other cycles, it needs to be measured within 3 days prior to chemotherapy; • If CBC is measured within 3 days prior to day 1 of cycle 1, it does not need to be measured again on day 1 of cycle 1. In other cycles, it needs to be measured within 3 days prior to chemotherapy; • Blood biochemistry does not need to be remeasured on day 1 of cycle 1 if it was performed within 3 days prior to day 1 of cycle 1. For other cycles, it needs to be measured within 3 days prior to chemotherapy. • If coagulation function has been measured within 3 days prior to day 1 of cycle 1, it does not need to be remeasured on day 1 of cycle 1. In other cycles, it needs to be measured within 3 days prior to chemotherapy; • If a urine test was performed within 3 days prior to day 1 of cycle 1, it does not need to be re-measured on day 1 of cycle 1. In other cycles, it needs to be measured within 3 days prior to chemotherapy; • Urine pregnancy test: Applicable only to women who may become pregnant. If performed within 3 days prior to day 1 of cycle 1, there is no need to retest on day 1 of cycle 1. In other cycles, it must be performed within 3 days prior to chemotherapy; • Rituximab administration; Day 2 Vital signs (respiration, blood pressure, pulse) should be measured once within 30 minutes before treatment, and again within 30 minutes before and 30 (±5) minutes after administration of f-bemarenograstim alfa. • A 12-lead electrocardiogram should be measured within 30 minutes prior to administration of efbemarenograstim alfa. Cyclophosphamide, 750 mg / m² 2 Intravenous infusion; doxorubicin, 50 mg / m² 2 Intravenous infusion; vincristine, 1.4 mg / m² 2 (Maximum 2.0 mg), administered intravenously. The above medication may be replaced with a similar medication as determined by the principal investigator; • Taking prednisone or prednisolone; Subcutaneous administration of efbemarenograstim alfa: Administered 4-6 hours after chemotherapy; Day 3 • Prednisone or prednisolone Days 4-21 • CBC Cycle 1: Blood samples are taken for hematological testing to monitor neutrophil counts on days 1, 6, 7, 8, 9, 10, and 14. ANC remains at 1.0 × 10⁶ on day 10. 9 If the ANC is below 1.0 × 10⁹ / L, monitor daily until the ANC recovers to 1.0 × 10⁹ / L or higher. Cycles 2-6: Blood samples for hematological testing are taken on days 1, 8, 10, and 14 to monitor neutrophil count. • Prednisone or prednisolone (taken on days 4, 5, and 6 of each cycle) Visit at the end of treatment (approximately 21 (+7) days after the last administration of the test drug) • 12-lead electrocardiogram examination; • For patients who complete treatment early, blood should be collected for CBC as specified in the cycle protocol to monitor changes in neutrophil count: ·Blood biochemistry; ·Blood clotting; • Urine test; • Urine pregnancy test.

[0248] Explanation of safety assessment Physical examination During the screening period, a complete physical examination will be conducted, including a general physical examination of the body, skin, lymph nodes, eyes, ears, nose, mouth, pharynx, neck, thyroid, chest, lungs, cardiovascular system, abdomen, limbs, musculoskeletal system, and any special examinations. A symptom-specific physical examination will be conducted at all other visits.

[0249] ECOG Performance Status Patient performance status is assessed using the ECOG criteria.

[0250] Vital signs assessment Systolic blood pressure, diastolic blood pressure, respiration, and pulse rate shall be recorded once during the screening period, within 30 minutes before treatment on day 1 of each cycle, within 30 minutes before treatment on day 1, within 30 minutes before and 30(±5) minutes after subcutaneous administration of efbemarenograstim alfa, and at the end of the study.

[0251] Body temperature Use a patient diary to record oral body temperature daily. Oral temperature should be measured within 30 minutes before treatment on the first and second days of each cycle.

[0252] If a participant's body temperature exceeds 38.0°C at any point, they must measure their temperature again after one hour. If the temperature remains above 38.0°C for more than one hour, the participant must contact the clinical trial site staff. If the temperature exceeds 38.3°C, the patient must immediately contact the clinical trial site staff.

[0253] 12-lead electrocardiogram (ECG) A 12-lead electrocardiogram includes heart rate, RR interval, PR interval, QT interval, QRS width, and QTc interval.

[0254] Test results from the past 7 days can be used as the result for day 1 of cycle 1. Tests for day 1 of other cycles must be performed within 3 days prior to chemotherapy. On day 2 of each cycle, a test must be performed within 30 minutes prior to administration of fbemarenograstim alfa.

[0255] Complete blood count (CBC) CBC testing includes red blood cell count, hemoglobin, white blood cell count, platelet count, white blood cell differential (neutrophils, lymphocytes, monocytes, basophils, and eosinophils), hematocrit, mean corpuscular hemoglobin level, mean corpuscular volume, and mean corpuscular hemoglobin concentration.

[0256] Cycle 1: Blood samples for hematological testing were taken on days 1, 6, 7, 8, 9, 10, and 14 to monitor neutrophil count. On day 10, the ANC was still 1.0 × 10⁶. 9If it is below / L, ANC is 1.0×10 9 Daily CBC is required until the neutrophil count recovers to above / L. Cycles 2-6: Blood samples for hematological testing are taken on days 1, 8, 10, and 14 to monitor neutrophil counts.

[0257] blood clotting Blood coagulation tests include prothrombin time, the International Standardized Index of prothrombin time and / or prothrombin activity, partially activated prothrombin time, and fibrinogen concentration D-dimer.

[0258] blood biochemistry Blood biochemistry tests include total protein, albumin, prealbumin, globulin, blood glucose, urea nitrogen, creatinine, alkaline phosphatase, lactate dehydrogenase, creatine kinase, blood amylase, total bilirubin, aspartate aminotransferase, alanine aminotransferase, ferritin, total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, calcium, phosphorus, magnesium, potassium, sodium, and chlorine.

[0259] Urine test A urine test includes pH, urine specific gravity, urobilinogen, latent blood, white blood cells, urine protein, urine glucose, bilirubin, ketones, urine red blood cells, and urine color.

[0260] Urine pregnancy test Urine pregnancy tests are used for female patients who may be pregnant.

[0261] In this study, subcutaneous administration of 20 mg of efbemarenograstim alfa 4-6 hours after R-CHOP-like chemotherapy in patients with non-Hodgkin lymphoma demonstrated favorable efficacy and an acceptable safety profile. This indicates that administering efbemarenograstim alfa on the same day as R-CHOP-like chemotherapy is effective and safe in lymphoma patients, offering patient benefits such as shorter hospital stays and a reduced risk of neutropenia. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6]

Claims

1. A method for treating or preventing a condition characterized by decreased leukocyte production in a human individual requiring treatment or prevention of such a condition, the method comprising administering an effective amount of granulocyte colony-stimulating factor (G-CSF) dimer to the human individual less than 24 hours after administering a chemotherapeutic agent or radiotherapy to the human individual.

2. The method according to claim 1, wherein the condition characterized by a decrease in leukocyte production includes one or more of the following: chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, decreased hematopoietic function, decreased immune function, decreased neutrophil count, decreased neutrophil recruitment, mobilization of peripheral blood progenitor cells, sepsis, infection, leukopenia, low bone marrow engraftment during transplantation, decreased bone marrow recovery in the treatment of myeloplasty or myelosuppression induced by radiation, chemicals or chemotherapy, radiotherapy-induced myeloplasty or myelosuppression, and acquired immunodeficiency syndrome.

3. The method according to claim 1 or 2, wherein the condition characterized by a decrease in leukocyte production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.

4. The method according to any one of claims 1 to 3, wherein the G-CSF dimer is administered within approximately 6 hours after the administration of the chemotherapeutic agent or the radiotherapy.

5. The method according to any one of claims 1 to 4, wherein the G-CSF dimer is administered within approximately 3 hours after the administration of the chemotherapeutic agent or the radiotherapy.

6. The method according to any one of claims 1 to 5, wherein the G-CSF dimer is administered within approximately two hours after the administration of the chemotherapeutic agent or the radiotherapy.

7. The method according to any one of claims 1 to 6, wherein the G-CSF dimer is administered within approximately 0.5 hours after the administration of the chemotherapeutic agent or the radiotherapy.

8. The method according to any one of claims 1 to 7, wherein the G-CSF dimer is administered simultaneously with the administration of the chemotherapeutic agent or the radiotherapy.

9. The method according to any one of claims 1 to 8, wherein the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.

10. The method according to claim 9, wherein the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.

11. The aforementioned human individual, i) Epirubicin and cyclophosphamide; ii) Docetaxel and cyclophosphamide; iii) Doxorubicin and cyclophosphamide; iv) Docetaxel and doxorubicin; v) Docetaxel, doxorubicin, and cyclophosphamide; or vi) Cyclophosphamide, doxorubicin, and vincristine, The method according to any one of claims 1 to 10, wherein two or more chemotherapeutic agents, including the above, are administered.

12. The method according to any one of claims 1 to 11, wherein the human individual is administered the chemotherapy agent or radiotherapy for treating cancer.

13. The method according to claim 12, wherein the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma.

14. The method according to any one of claims 1 to 13, wherein the human individual is administered the chemotherapeutic agent or radiotherapy for at least four cycles, and the G-CSF dimer is administered in cycle 1 less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy.

15. The method according to claim 14, further comprising administering an effective amount of G-CSF dimer about 24 hours after the administration of the chemotherapeutic agent or radiotherapy in at least each of the 2 to 4 cycles.

16. The method according to any one of claims 1 to 14, wherein the human individual is administered the chemotherapeutic agent or radiotherapy for at least four cycles, and the G-CSF dimer is administered in each of the at least four cycles less than 24 hours after the administration of the chemotherapeutic agent or radiotherapy.

17. The method according to any one of claims 14 to 16, wherein each cycle is approximately 21 days.

18. The method according to any one of claims 1 to 17, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising a G-CSF monomer and a dimerizing domain.

19. The method according to claim 18, wherein the G-CSF monomer comprises the amino acid sequence of SEQ ID NO:

1.

20. The method according to claim 18 or 19, wherein within each monomer subunit, the G-CSF monomer is connected to the dimerization domain via an arbitrary linker.

21. The method according to claim 20, wherein the linker has a length of about 6 to about 30 amino acids.

22. The method according to claim 20 or 21, wherein the linker comprises the amino acid sequence of SEQ ID NO:

12.

23. The method according to any one of claims 18 to 22, wherein the dimerization domain within each monomer subunit contains at least two cysteine ​​molecules capable of forming intermolecular disulfide bonds.

24. The method according to any one of claims 18 to 23, wherein the dimerizing domain comprises at least a portion of the Fc fragment.

25. The method according to claim 24, wherein the Fc fragment comprises CH2 and CH3 domains.

26. The method according to claim 24 or 25, wherein the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or a fragment or variant thereof.

27. The method according to any one of claims 24 to 26, wherein the Fc fragment comprises the amino acid sequence of any one of sequence numbers 2 to 5.

28. The method according to any one of claims 18 to 27, wherein the G-CSF monomer is located on the C-terminal side of the dimerization domain within each monomer subunit.

29. The method according to any one of claims 18 to 27, wherein the G-CSF monomer is located on the N-terminal side of the dimerization domain within each monomer subunit.

30. The method according to one of claims 18 to 27 and 29, wherein each monomeric subunit comprises the amino acid sequence of any of SEQ ID NOs: 6 to 10, or a variant thereof having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs: 6 to 10.

31. The method according to one of claims 18 to 27, 29, and 30, wherein each monomer subunit contains the amino acid sequence of any of SEQ ID NOs: 6 to 10.

32. The method according to one of claims 18 to 27, 29 to 31, wherein each monomer subunit contains the amino acid sequence of SEQ ID NO:

6.

33. The method according to any one of claims 1 to 27, 29 to 32, wherein the G-CSF dimer is efbemalenograstim alpha.

34. The G-CSF dimer is formulated into a pharmaceutical composition, and the pharmaceutical composition is (a) G-CSF dimer at approximately 20 mg / mL, (b) Approximately 10 mM sodium acetate and (c) EDTA of approximately 1 mM, (d) Approximately 5% (w / v) sorbitol and (e) Contains approximately 0.01% (w / v) of polysorbate 20, The pharmaceutical composition having a pH of about 5.2, according to the method according to any one of claims 1 to 33.

35. The method according to claim 34, wherein the pharmaceutical composition is contained in a syringe.

36. The method according to claim 35, wherein the volume of the pharmaceutical composition in the syringe is approximately 1 mL.

37. The method according to claim 35 or 36, wherein the syringe is for single use.

38. The method according to any one of claims 35 to 37, wherein the syringe is sterilized.

39. The method according to any one of claims 1 to 38, wherein the G-CSF dimer is administered in an amount of about 5 mg to about 25 mg per dose.

40. The method according to any one of claims 1 to 39, wherein the G-CSF dimer is administered in an amount of about 10 mg to about 25 mg per dose.

41. The method according to any one of claims 1 to 40, wherein the G-CSF dimer is administered at a dose of approximately 20 mg per administration.

42. The method according to any one of claims 1 to 41, wherein the G-CSF dimer is administered subcutaneously.

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